CN110680949B

CN110680949B - Preparation method and application of wound dressing based on breast milk

Info

Publication number: CN110680949B
Application number: CN201910994497.6A
Authority: CN
Inventors: 吴钧; 冼彩虹; 顾志鹏; 刘桂廷
Original assignee: Sun Yat Sen University
Current assignee: Sun Yat Sen University
Priority date: 2019-10-18
Filing date: 2019-10-18
Publication date: 2021-06-29
Anticipated expiration: 2039-10-18
Also published as: CN110680949A

Abstract

The invention discloses a preparation method and application of a wound dressing based on breast milk. The invention provides a preparation method of a wound dressing based on breast milk, which comprises the following steps: s1, freeze-drying breast milk, and dissolving the breast milk in a biocompatible solvent to obtain a breast milk solution; wherein the concentration of the breast milk solution in the step S1 is more than or equal to 1 mug/mL; s2, filtering and sterilizing the breast milk solution obtained in the step S1 to obtain the wound dressing. The wound dressing has low immunogenicity, and can not cause the body to generate additional inflammatory reaction; can eliminate hydroxyl free radicals and active oxygen in cells, improve the proliferation rate of the cells, promote the migration of fibroblasts and endothelial cells, and enhance the hemangiogenic capability of the endothelial cells; regulating macrophage M2 phenotype, promoting hair follicle regeneration, and normalizing healing wound. In addition, the material of the wound dressing is derived from food breast milk, is green and safe, has simple and convenient preparation process, and has wide popularization and application prospect.

Description

Preparation method and application of wound dressing based on breast milk

Technical Field

The invention belongs to the technical field of biological medicines. And more particularly, to a method of making and using a breast milk-based wound dressing.

Background

The skin is the largest organ of the human body and its functions include resistance to microorganisms and regulation of body temperature. Thus, once the skin is injured, it is necessary to accelerate wound healing rapidly to avoid further formation of chronic wounds. More importantly, skin repair has a considerable drawback in that severely damaged skin can heal, but cannot regenerate, and scarring can occur; the formation of these scars is not only a defect in appearance, but also hinders the normal movement of the human body, and prevents the body from cooling due to the lack of sweat glands, resulting in the increase of the body temperature and metabolic disorders of the human body. Although scars appear thicker than normal skin, in practice they are less elastic than normal skin, and severely impede the normal function of the skin tissue. Fortunately, once a hair follicle has grown in a skin wound, the skin tends to normalize. Therefore, promoting skin tissue reconstruction and hair follicle regeneration at the wound site while accelerating wound healing plays a crucial role in promoting wound repair and reducing scar tissue formation.

In the existing medicines for treating wounds, western medicines are usually medicines with the functions of diminishing inflammation, resisting bacteria, relieving pain and the like, such as antibiotics, silver salts and the like, and mainly take the effects of diminishing inflammation, relieving pain and resisting bacteria; but has the defects of large side effect, poor capability of promoting skin tissue regeneration on deep wound surfaces, unsatisfactory scar repair effect, high price and the like, so the clinical application of the wound healing ointment has certain limitation. The traditional Chinese medicine is wound ointment with the functions of diminishing inflammation, promoting granulation, removing heat toxin and the like, such as Jingwanhong, Mei wound ointment, Bao moisture burn ointment and the like; however, they have many disadvantages, such as a secret recipe handed down from ancestors or a clinical proved recipe, and have a long history of use, and the curative effect is demonstrated mainly by the experience of medication and lack of systematic pharmacological research experimental data. At present, some medicines for treating scars, such as hormone medicines, anti-tumor medicines, P substance inhibitors, cytokines, immunosuppressants and the like, are available in the market; however, these drugs are expensive, complicated in preparation process, inconsistent in effect, and have adverse reactions to a certain extent.

Thus, there is a need to develop more drugs or biomaterials to provide a more effective approach to promote wound repair and reduce scar tissue formation.

Disclosure of Invention

The invention aims to solve the technical problem of overcoming the defects and shortcomings of the existing medicine for treating the wound and provides a wound dressing based on breast milk and a preparation method and application thereof.

The invention researches the influence of the wound dressing based on breast milk on three stages (an inflammation stage, a proliferation stage and a remodeling stage) of wound repair, finds that the wound dressing has lower immunogenicity, does not additionally aggravate inflammatory reaction, and has good biocompatibility; in the inflammatory phase of wound repair, the composition can promote endothelial cell migration, has antioxidant property, can remove active oxygen generated by free radicals and cell stress, provides support for accelerating healing, can adjust M2 type macrophage phenotype, promotes later-stage hair follicle formation, and plays a positive role in inhibiting the formation of scar tissues; in the proliferation stage, the proliferation rate of oxidative stress cells caused by the inflammation stage can be improved, the migration of fibroblasts and the formation of blood vessels are promoted, and the promotion of wound healing is facilitated; in the remodeling stage, the synthesis of collagen and glycosaminoglycan is promoted, the preparation is made for structural remodeling, and the transformation of macrophages to M2 type is promoted, so that formed collagen fibers are arranged regularly, the wound tissue repair is promoted, and the formation of scars is inhibited or reduced.

It is an object of the present invention to provide a breast milk based wound dressing.

It is another object of the present invention to provide a method of preparing the breast milk-based wound dressing.

It is a further object of the present invention to provide the use of the wound dressing as, or in the manufacture of, a product or article for promoting wound repair and/or reducing scar tissue formation.

The above purpose of the invention is realized by the following technical scheme:

the invention firstly provides a wound dressing based on breast milk, which comprises the following steps:

s1, freeze-drying breast milk, and dissolving the breast milk in a biocompatible solvent to obtain a breast milk solution; wherein the concentration of the breast milk solution in the step S1 is more than or equal to 1 mug/mL;

s2, filtering and sterilizing the breast milk solution obtained in the step S1 to obtain the wound dressing.

Preferably, in step S2, the breast milk solution obtained in step S1 is filtered, added to a biocompatible matrix, and sterilized.

Preferably, the concentration of the breast milk solution in the step S1 is 1-10000 mug/mL.

More preferably, the concentration of the breast milk solution in the step S1 is 10-1000 [ mu ] g/mL.

Still more preferably, the concentration of the breast milk solution in step S1 is 100-500. mu.g/mL.

Still further preferably, the concentration of the breast milk solution of step S1 is 500. mu.g/mL.

Preferably, the freeze-drying time of the step S1 is 2-8 d.

When the freeze-drying time is too long (more than 8 days), the efficiency is low; when the time for lyophilization is too short (less than 2d), the breast milk solution cannot be completely lyophilized into a powder.

More preferably, the freeze-drying time of step S1 is 5 d.

Preferably, the temperature of the lyophilization in the step S1 is-50 ℃ to-30 ℃.

When the freeze-drying temperature is too high (more than-30 ℃), the freeze-drying effect is poor, and longer freeze-drying time is needed; when the temperature for lyophilization is too low (less than-50 ℃), the minimum lyophilization temperature of the lyophilizer is limited by its own performance.

More preferably, the temperature of the lyophilization in step S1 is-45 ℃.

Preferably, the biocompatible solvent in step S1 is selected from one or more of water, PBS buffer or liquid culture medium.

The source of the water is not particularly limited and may be tap water, deionized water, distilled water, ultrapure water or the like.

More preferably, the pH value of the PBS buffer solution is 6.5-7.5.

Even more preferably, the PBS buffer has a pH of 7.

More preferably, the liquid culture medium is selected from one or more of DMEM liquid culture medium, MEM liquid culture medium, RPMI 1640 liquid culture medium, F12 liquid culture medium or IMDM liquid culture medium.

Preferably, the substrate in step S2 is one or more of medical gauze, medical sponge, medical foam or hydrogel.

More preferably, the hydrogel is a hydrogel formed by physical action, such as a gel formed by blending polyacrylamide with polyacrylic acid, or the like; gel formed by interaction of water-soluble polymer and metal ion, such as sodium alginate and chitosan, which respectively form sodium alginate, chitosan hydrogel and the like with calcium ion and phosphate radical; hydrogel formed by interaction of polycation electrolyte and polyanion electrolyte, such as alginic acid-chitosan hydrogel formed by interaction of alginic acid and chitosan; the polymer chains polymerize to form hydrogels such as gelatin and agar.

Even more preferably, the hydrogel is Pluronic (Pluronic F-127).

Preferably, the step S2 is performed by performing filter sterilization with a needle sterilization filter.

More preferably, the filter head of the needle sterilizing filter is a micropore sterilizing filter membrane with the diameter of 0.1-0.8 mu m.

Still more preferably, the filter head of the needle sterilization filter is a 0.22 μm micro-porous sterilization filter membrane.

In addition, the invention also provides the wound dressing based on breast milk prepared by the method.

The use of the wound dressing as or in the manufacture of a product or article for promoting wound repair and/or reducing scar tissue formation is also within the scope of the invention.

Preferably, the application refers to that the wound dressing can eliminate hydroxyl free radicals and active oxygen in cells and improve the proliferation rate of the cells.

Preferably, the application refers to that the wound dressing can promote the migration of fibroblasts and endothelial cells and enhance the angioblasts of the endothelial cells.

Preferably, the application means that the wound dressing is capable of modulating the M2 phenotype of macrophages, promoting hair follicle regeneration, reducing scar tissue formation, and normalizing healing wounds.

The invention has the following beneficial effects:

the invention provides a preparation method and application of a wound dressing based on breast milk. The wound dressing based on breast milk prepared by the invention has lower immunogenicity, does not aggravate inflammatory reaction additionally, and has good biocompatibility; in the inflammatory phase of wound repair, the composition can promote endothelial cell migration, has antioxidant property, can remove active oxygen generated by free radicals and cell stress, provides support for accelerating healing, can adjust M2 type macrophage phenotype, promotes later-stage hair follicle formation, and plays a positive role in inhibiting the formation of scar tissues; in the proliferation stage, the proliferation rate of oxidative stress cells caused by the inflammation stage can be improved, the migration of fibroblasts and the formation of blood vessels are promoted, and the promotion of wound healing is facilitated; in the remodeling stage, the synthesis of collagen and glycosaminoglycan is promoted, and preparation is made for structural remodeling; also can promote the macrophage to transform to M2 type, arrange the formed collagen fiber regularly, promote wound tissue repair, and inhibit or reduce scar formation.

In addition, the material of the wound dressing is derived from food breast milk, and the preparation process is simple, green and safe and is easy to realize; therefore, the wound dressing provided by the invention has wide popularization and application prospects in serving as or preparing products or products for promoting wound repair and/or reducing scar tissue formation.

Drawings

FIG. 1 shows the results of the detection of the expression of the immunogenic cytokine TNF- α in different concentrations of breast milk.

FIG. 2 shows the results of hydroxyl radical scavenging performance tests of breast milk solutions of different concentrations.

FIG. 3 is a graph showing the effect of different concentrations of breast milk solution on scavenging reactive oxygen species from cells.

FIG. 4 is a graph showing the effect of different concentrations of breast milk on the rate of cell proliferation under oxidative conditions.

FIG. 5 shows the effect of different concentrations of breast milk on endothelial cell migration.

FIG. 6 is a graph of the quantification of the effect of different concentrations of breast milk solution on endothelial cell migration.

FIG. 7 shows the effect of different concentrations of breast milk on fibroblast migration.

FIG. 8 is a graph of the quantification of the effect of different concentrations of breast milk solution on fibroblast migration.

FIG. 9 shows the effect of different concentrations of breast milk solution on the vascularization ability of cells.

FIG. 10 is a graph of the effect of different concentrations of breast milk on the macrophage M2 phenotype.

FIG. 11 is a graph of the results of HE staining, Masson staining and immunohistochemical staining of the effects of breast milk-based wound dressings of the present invention on rat skin dorsal wound tissue repair.

Detailed Description

The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

Example 1 preparation of breast milk-based wound dressing

A method of preparing a breast milk-based wound dressing, comprising the steps of:

s1, freeze-drying breast milk at the freeze-drying temperature of-45 ℃ for 5d, and dissolving the freeze-dried breast milk in PBS to obtain a breast milk solution;

s2, filtering and sterilizing the breast milk solution obtained in the step S1 by using a needle sterilizing filter of a micropore sterilizing filter membrane with a filter head of 0.22 mu m, and adsorbing a supernatant into Pluronic F-127 matrix to obtain the wound dressing; wherein the concentration of the breast milk solution in the step S1 is 500 mug/mL.

EXAMPLE 2 preparation of Breast milk-based wound dressing

s1, freeze-drying breast milk at the freeze-drying temperature of-50 ℃ for 2d, and dissolving the freeze-dried breast milk in water to obtain a breast milk solution;

s2, filtering and sterilizing the breast milk solution obtained in the step S1 by using a needle sterilizing filter of a micropore sterilizing filter membrane with a filter head of 0.1 mu m, adsorbing supernatant into gauze, and sterilizing to obtain the wound dressing; wherein the concentration of the breast milk solution in the step S1 is 1 mug/mL.

Example 3 preparation of breast milk-based wound dressing

s1, freeze-drying breast milk at the freeze-drying temperature of-30 ℃ for 8d, and dissolving the freeze-dried breast milk in a DMEM liquid culture medium to obtain a breast milk solution;

s2, filtering and sterilizing the breast milk solution obtained in the step S1 by using a needle sterilizing filter of a micropore sterilizing filter membrane with a filter head of 0.8 mu m, adsorbing a supernatant into a sponge, and sterilizing to obtain the wound dressing; wherein the concentration of the breast milk solution in the step S1 is 10 mug/mL.

Example 4 preparation of breast milk-based wound dressing

s1, freeze-drying breast milk at the freeze-drying temperature of-48 ℃ for 7d, and dissolving the freeze-dried breast milk in an MEM liquid culture medium to obtain a breast milk solution;

s2, filtering and sterilizing the breast milk solution obtained in the step S1 by using a needle sterilizing filter of a micropore sterilizing filter membrane with a filter head of 0.3 mu m, adsorbing supernatant into foam, and sterilizing to obtain the wound dressing; wherein the concentration of the breast milk solution in the step S1 is 100 mug/mL.

Example 5 preparation of breast milk-based wound dressing

s1, freeze-drying breast milk at the freeze-drying temperature of-32 ℃ for 3d, and dissolving the freeze-dried breast milk in an RPMI 1640 liquid culture medium to obtain a breast milk solution;

s2, filtering and sterilizing the breast milk solution obtained in the step S1 by using a needle sterilizing filter of a micropore sterilizing filter membrane with a filter head of 0.5 mu m, adsorbing supernatant into gauze, and sterilizing to obtain the wound dressing; wherein the concentration of the breast milk solution in the step S1 is 500 mug/mL.

Example 6 preparation of breast milk-based wound dressing

s1, freeze-drying breast milk at the freeze-drying temperature of-30 ℃ for 8d, and dissolving the freeze-dried breast milk in an F12 liquid culture medium to obtain a breast milk solution;

s2, filtering and sterilizing the breast milk solution obtained in the step S1 by using a needle sterilizing filter of a micropore sterilizing filter membrane with a filter head of 0.7 mu m, adsorbing a supernatant into a sponge, and sterilizing to obtain the wound dressing; wherein the concentration of the breast milk solution in the step S1 is 1000 mug/mL.

Example 7 preparation of breast milk-based wound dressing

s1, freeze-drying breast milk at the freeze-drying temperature of-45 ℃ for 5d, and dissolving the freeze-dried breast milk in an IMDM liquid culture medium to obtain a breast milk solution;

s2, filtering and sterilizing the breast milk solution obtained in the step S1 by using a needle sterilizing filter of a micropore sterilizing filter membrane with a filter head of 0.6 mu m, adsorbing supernatant into foam, and sterilizing to obtain the wound dressing; wherein, the concentration of the breast milk solution in the step S1 is 10000 mug/mL.

Application example 1 testing of the expression of the immunogenic cytokine TNF-alpha in Breast milk solution

1. Experimental methods

(a) Inoculating RAW 264.7 macrophage in a 12-hole plate, and adding a culture medium containing serum to be attached to the wall;

(b) removing old culture medium, adding 5 serum culture media containing different concentrations (10 μ g/mL, 50 μ g/mL, 100 μ g/mL, 300 μ g/mL and 500 μ g/mL) of breast milk solution, respectively, culturing for 1 day, and adding serum-containing culture medium to control group;

(c) removing the old culture medium, washing the cells for 2 times by using a PBS solution, adding 1mL of a TRIZOL solution into each sample, uniformly blowing, and standing in a 1.5mL EP tube at room temperature for 5 min;

(d) adding 200 μ L chloroform into each tube, shaking vigorously for 15s, standing at room temperature for 3min, 4 deg.C, 14000g, and centrifuging for 15 min; taking 200-400 mu L of supernatant in a new EP tube, adding 0.5mL of isopropanol into each tube, standing at room temperature for 10min, standing at 4 ℃, 14000g, and centrifuging for 15 min; discarding the supernatant, adding ice-precooled 75% ethanol (DEPC water), washing for 2 times, 12000g, and centrifuging for 5 min; discarding the supernatant, naturally drying at room temperature for 5-10 min, and dissolving RNA by using 20 mu LDEPC water;

(e) detecting the purity and concentration of RNA by using a nucleic acid protein detector;

(f) taking 1 mu g of RNA for reverse transcription in each 20 mu L system;

(g) and (3) carrying out qRT-PCR reaction by using a PCR instrument, and detecting the expression condition of the cell factor TNF-alpha.

2. Results of the experiment

The results of the effect of the breast milk solutions with different concentrations on the expression of the cytokine TNF-alpha are shown in fig. 1, and it can be seen that after the RAW 264.7 macrophage is incubated in the environment of the breast milk solutions with different concentrations for 1 day, compared with the control group, the expression of the TNF-alpha is not different; when the concentration of the breast milk solution is lower than 300 mug/mL, the expression of TNF-alpha is less than that of a control group, but no obvious difference exists; when the concentration of the breast milk solution is more than or equal to 300 mug/mL, the expression of TNF-alpha is more than that of a control group, but the expression is not significantly different. The above results indicate that the breast milk solution does not stimulate cells to produce inflammatory reactions and has low immunogenicity.

Application example 2 hydroxy radical scavenging Performance test of Breast milk solution

1. Experimental methods

(a) Preparation of 8mM FeSO₄Solution (dissolved in ultrapure water), 2mM H₂O₂(diluted with ultrapure water), 4mM sodium salicylate solution (dissolved in absolute ethanol), and 5 breast milk solutions (diluted by immersion in ultrapure water) of different concentrations (10. mu.g/mL, 50. mu.g/mL, 100. mu.g/mL, 300. mu.g/mL, and 500. mu.g/mL);

(b) experimental group At: to 200. mu.L of FeSO₄The solution was added 200. mu.L of 5 different concentrations of breast milk solution, 200. mu. L H₂O₂Shaking and standing the solution and 200 μ L sodium salicylate solution for 30 min;

blank group Ac: to 200. mu.L of FeSO₄Adding 200 μ L breast milk solution, 200 μ L ultrapure water and 200 μ L sodium salicylate solution into the solution, and shaking and standing for 30 min;

background group Ab: to 200. mu.L of FeSO ₄200. mu.L of ultrapure water and 200. mu. L H were added to the solution₂O₂The solution and 200. mu.L of sodium salicylate solution were shaken and left to stand for 30 min.

(c) Respectively taking 200 mu L of mixed liquor from each sample of the experimental group At, the blank group Ac and the background group Ab to a 96-well plate, setting the wavelength of an enzyme-labeling instrument to be 510nm, determining the light absorption value of each well, and according to a formula: AA [% ], [1- (At-Ac)/Ab ] × 100%, and the clearance rate was calculated.

2. Results of the experiment

The results of the hydroxyl radical scavenging performance test of the breast milk solutions with different concentrations are shown in fig. 2, and it can be seen that the hydroxyl radical scavenging rate is continuously improved along with the increase of the concentration of the breast milk solution; the concentration of the breast milk solution was 10. mu.g/mL, 50. mu.g/mL, 100. mu.g/mL, 300. mu.g/mL and 500. mu.g/mL, respectively, and the clearance rates of the corresponding hydroxyl radicals were 9.5%, 12.6%, 17.7%, 22% and 32%, respectively, showing a concentration-dependent characteristic.

Application example 3 Effect of breast milk solution on scavenging intracellular reactive oxygen species

1. Experimental methods

(a) NIH 3T3 cells (mouse embryo fibroblasts) are inoculated in a 24-well plate, and a culture medium containing serum is added for adherence;

(b) removing old culture medium, adding 5 breast milk solutions with different concentrations (10 μ g/mL, 50 μ g/mL, 100 μ g/mL, 300 μ g/mL and 500 μ g/mL), culturing for 24 hr, and adding culture medium containing serum to positive control group and negative control group;

(c) removing old culture medium, adding 5 breast milk solutions with different concentrations and H₂O₂The solution (5 mu g/mL) is cultured for 24H, and the positive control group is added with a culture medium containing serum and H₂O₂Solution (5. mu.g/mL), negative control group was added with serum-containing medium and H₂O₂PBS solution with the same solution volume;

(d) removing the old culture medium, washing the cells for 2 times by using a PBS solution, and incubating 300 mu L of DCFH-DA probe for 20min in each hole;

(e) the probe solution was removed, the cells were washed 2 times with PBS solution, and the well plate was placed under an inverted fluorescence microscope to observe the fluorescence intensity.

2. Results of the experiment

The scavenging effect of the breast milk solution with different concentrations on Reactive Oxygen Species (ROS) in cells is shown in fig. 3, and it can be seen that the breast milk solution has remarkable capacity of scavenging the reactive oxygen species in cells, and the scavenging rate presents the characteristic of concentration dependence; wherein, fig. (1) shows that intracellular ROS levels are lower in the absence of any stimulation of the cells (negative control); cells were incubated with 5. mu.g/mL H₂O₂After pretreatment with solution (positive control), cellsA significant increase in internal ROS levels; intense fluorescence can be observed in FIG. 2; however, as can be seen from FIGS. 3 to 5, the fluorescence intensity of the cells was significantly reduced in the order of 10. mu.g/mL, 50. mu.g/mL and 100. mu.g/mL of breast milk solution pretreatment; when the cells are pretreated with a breast milk solution having a concentration of 100. mu.g/mL or more, little fluorescence is found in the cells, i.e., there is substantially no ROS in the cells. The results show that the breast milk solution has good intracellular antioxidant activity, and the result of scavenging the intracellular active oxygen is consistent with the result of scavenging the free radicals.

Application example 4 Effect of Breast milk solution on cell proliferation Rate under oxidative conditions

1. Experimental methods

(a) NIH 3T3 cells (mouse embryo fibroblasts) were seeded in 96-well plates (5000/well), and serum-containing medium was added overnight to allow adherence;

(b) removing old culture medium, adding 5 serum culture mediums containing breast milk solution with different concentrations (10. mu.g/mL, 50. mu.g/mL, 100. mu.g/mL, 300. mu.g/mL and 500. mu.g/mL) into experimental group, adding serum culture mediums into positive control group and negative control group, and culturing for 24 h;

(c) old medium was removed and 100. mu.L of 5 serum media containing breast milk solution of different concentrations and H were added to each well₂O₂Solution (5. mu.g/mL), positive control group was added serum-containing medium and H₂O₂Solution (5. mu.g/mL), negative control group was added with serum-containing medium and H₂O₂Setting 5 multiple holes in PBS solution with the same solution volume;

(d) after co-culturing for 24h, 48h and 96h, adding 20 mu L of MTT solution into each hole, and incubating for 4 h;

(e) removing the culture solution, adding 100 mu L DMSO into each hole to dissolve crystals, oscillating for 8-10 min by a shaking table, placing the hole plate on an enzyme-labeling instrument, and detecting the light absorption value by adopting the wavelength of 490 nm.

2. Results of the experiment

The results of the effect of different concentrations of breast milk solution on the cell proliferation rate under the oxidizing condition are shown in fig. 4, and it can be seen that the cell proliferation rate is greatly reduced when the cells are in the oxidant environment; however, after pretreatment with a breast milk solution, the cell growth rate tends to increase, and the cell growth rate increases with the increase in the concentration of the breast milk solution, and is characterized by concentration dependence.

Application example 5 Effect of different concentrations of Breast milk solution on endothelial cell and fibroblast migration

1. Experimental methods

(a) Planking, 6-well plates (25X 10 holes per well)⁴) When the cells are full;

(b) marking 3 straight lines on each pore plate by using a 200uL gun head, removing old liquid, gently cleaning the pore plate by using PBS (phosphate buffer solution) for 1-2 times, adding a serum-free culture medium, photographing, and then putting the culture medium back into an incubator for culture;

(c) and observing the migration condition of the scratches every 12h, photographing, and finishing the experiment after the migration of a certain group is finished.

2. Results of the experiment

The results of the effect of different concentrations of breast milk on endothelial cell migration are shown in FIG. 5, and it can be seen that 10. mu.g/mL, 50. mu.g/mL, 100. mu.g/mL, 300. mu.g/mL and 500. mu.g/mL of breast milk can promote endothelial cell migration and also exhibit concentration-dependent characteristics. The quantitative results of the effect of different concentrations of breast milk solution on the endothelial cell migration rate are shown in fig. 6, and it can be seen that after 12h, the endothelial cell migration rates were 61%, 62%, 66%, 82% and 78%, respectively, while the control group was only 38%; after 24h, the endothelial cell migration rates were 85%, 84%, 88%, 98% and 100%, respectively, while the control group was only 60%, which showed significant differences compared to the control group by calculation. This result indicates that the breast milk solution promotes endothelial cell migration.

The effect of different concentrations of breast milk on fibroblast migration results are shown in FIG. 7, and it can be seen that 10. mu.g/mL, 50. mu.g/mL, 100. mu.g/mL, 300. mu.g/mL and 500. mu.g/mL breast milk solutions all promote fibroblast migration. The quantitative results of the effect of different concentrations of breast milk solutions on the fibroblast migration rate are shown in fig. 8, and it can be seen from fig. 8 that after 12h from low to high concentration, the fibroblast migration rates are 52%, 48%, 46%, 66% and 60%, respectively, while the control group is only 31%; after 24h, the migration rates of the fibroblasts were 88%, 95%, 94%, 98% and 96%, respectively, while the control group was only 76%, which showed significant differences compared with the control group by calculation. This result indicates that the breast milk solution promotes the migration of fibroblasts.

Application example 6 Effect of different concentrations of Breast milk solution on the vascularization ability of cells

1. Experimental methods

(a) Thawing matrigel at 4 ℃, and adding 10uL matrigel on an ibidi 96-well plate after the matrigel is thawed;

(b) placing in 37 deg.C incubator for solidification for 30 min;

(c) at the same time, 1X 10 was prepared⁴50uL of cell suspension;

(d) after 30min, 50uL of cell suspension was added to each well;

(e) after 30min, the cells were completely settled on matrigel, the culture solution was gently aspirated, and the medium containing the material was added separately;

(f) after 2h, 4h, 6h, 8h and 10h, the tube formation is observed and photographed.

2. Results of the experiment

The results of the influence of the breast milk solutions with different concentrations on the vascularization ability of cells are shown in fig. 9, and it can be seen that the breast milk solutions can not only promote the formation of the lumen structure, but also promote the formation of the number of the channels; compared with a control group, the breast milk solution group can form an obvious cavity after 2 hours, and can form a tubular structure earlier; and along with the migration of time, the tubular structure becomes more and more obvious after 4h, and the number of pipelines becomes more and more. The above results show that the breast milk solution can promote the formation of the cavity structure and the formation of the number of the pipelines, and the breast milk solutions with different concentrations do not have great difference on the formation of the number of the pipelines.

Application example 7 Effect of different concentrations of Breast milk solution on the macrophage M2 phenotype

The M2 type macrophage secretes IL-10 cytokine, so the expression of the IL-10 cytokine proves the phenotype of the macrophage M2, and the specific experimental method and the experimental result are as follows:

1. experimental methods

(b) removing old culture medium, adding 5 serum culture media containing different concentrations of breast milk solution, culturing for 3 days, and adding serum culture medium into control group;

(c) removing old culture medium, washing cells for 2 times with PBS solution, adding 1mL of TRIZOL solution into each sample, blowing and beating uniformly, and standing in 1.5mL of EP tube at room temperature for 5 min;

(f) taking 1 mu g of RNA for reverse transcription in each 20 mu L system;

(g) and (3) carrying out qRT-PCR reaction by using a PCR instrument, and detecting the expression condition of the cytokine IL-10.

2. Results of the experiment

The effect results of different concentrations of breast milk solutions on the phenotype of macrophage M2 are shown in FIG. 10, and it can be seen that after incubation of RAW 264.7 macrophage in the environment of different concentrations of breast milk solutions for 3 days, there is no difference in IL-10 expression after pretreatment of breast milk solutions with concentrations of 10 μ g/mL and 50 μ g/mL compared with the control group; when the concentration of the breast milk solution is increased to 100 mug/mL, the expression of IL-10 is obviously increased; when the concentration of the breast milk solution is increased to 500 mug/mL, the IL-10 expression continues to be significantly increased; indicating the conversion of macrophages to type M2 at a concentration of 100. mu.g/mL and 500. mu.g/mL in breast milk solution. The above results indicate that when the concentration of breast milk solution is greater than or equal to 100 μ g/mL, IL-10 expression can be significantly promoted.

Application example 8 impact of breast milk-based wound dressing on wound repair

1. Experimental methods

(1) Skin wound modeling

(a) Selecting male SD rats with the weight of 200-230 g as study objects, and randomly dividing all mice into two groups;

(b) all rats were shaved on their backs, anesthetized by intraperitoneal injection of chloral hydrate (10 wt%, 0.3mL/100g), and the 1.5cm circular skin on the rat back was excised all over;

(2) preparation of breast milk-based wound dressings

(a) 20g of Pluronic F127 matrix was weighed and dissolved in 80g of PBS and stirred overnight at 4 ℃;

(b) weighing a certain mass of breast milk powder according to the mass-to-volume ratio, dissolving the breast milk powder into Pluronic F127 matrix to enable the concentration of breast milk in the matrix to be 100 mu g/mL, and gelatinizing at room temperature to obtain the wound dressing based on breast milk.

(3) Wound medicine application

The control group was applied with a commercial dressing (3M company) to the wound, and the experimental group was applied with 100. mu.g/mL of the breast milk-based wound dressing prepared according to the present invention, and the wound was applied with the drug every day and photographed.

2. Results of the experiment

The HE dyeing, Masson dyeing and immunohistochemical dyeing results of the wound dressing based on breast milk, which is prepared by the invention, on the repairing influence of the wound surface tissues on the back of the skin of a rat are shown in fig. 11, and it can be seen that the wound dressing based on breast milk can promote the wound to form richer blood vessels, so that collagen fibers are gathered and arranged in parallel, and the formation of hair follicles of skin appendages can be promoted; as can be seen from the results of HE staining and Masson staining, compared with the control group, the wound granulation tissue degree of the breast milk-based wound dressing group is higher, the blood vessel distribution is rich, abundant hair follicles appear, and the structure is very similar to that of normal skin; from immunohistochemical staining results, it can be seen that the breast milk-based wound dressing group increased the number of blood vessels, promoted capillary neogenesis, improved blood vessel microcirculation, facilitated wound healing and reduced scar tissue formation compared to the control group.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. A method for preparing a breast milk-based wound dressing, comprising the steps of:

s1, freeze-drying breast milk, and dissolving the breast milk in a biocompatible solvent to obtain a breast milk solution;

s2, filtering and sterilizing the breast milk solution obtained in the step S1 to obtain the wound dressing;

in step S2, filtering the breast milk solution obtained in step S1, adding the filtered breast milk solution into a biocompatible matrix, and sterilizing to obtain the breast milk;

the concentration of the breast milk solution in the step S1 is 1-10000 mug/mL; the freeze-drying time of the step S1 is 2-8 days; the freeze-drying temperature is-50 ℃ to-30 ℃;

step S1, the biocompatible solvent is selected from one or more of water, PBS buffer solution or liquid culture medium, and the water is one or more of tap water, deionized water, distilled water or ultrapure water; the pH value of the PBS buffer solution is 6.5-7.5; the liquid culture medium is selected from one or more of DMEM liquid culture medium, MEM liquid culture medium, RPMI 1640 liquid culture medium, F12 liquid culture medium or IMDM liquid culture medium.

2. The method according to claim 1, wherein the biocompatible matrix in step S2 is one or more selected from medical gauze, medical sponge, medical foam, and hydrogel.

3. The method according to claim 1, wherein the step S2 is performed by filtering and sterilizing with a needle sterilizing filter; the filter head of the needle sterilizing filter is a micropore sterilizing filter membrane with the diameter of 0.1-0.8 mu m.

4. A breast milk-based wound dressing prepared by the method of any one of claims 1 to 3.

5. Use of the wound dressing of claim 4 as or in the manufacture of a product or article for promoting wound repair and/or reducing scar tissue formation.