CN105013013B

CN105013013B - Preparation method of skin ulcer repairing matrix

Info

Publication number: CN105013013B
Application number: CN201510454579.3A
Authority: CN
Inventors: 姚远
Original assignee: Guangdong Bo And Regenerative Medicine Co Ltd
Current assignee: Guangdong Bo And Regenerative Medicine Co Ltd
Priority date: 2015-07-29
Filing date: 2015-07-29
Publication date: 2020-01-21
Anticipated expiration: 2035-07-29
Also published as: CN110975011B; CN110975011A; CN110923193A; CN105013013A

Abstract

The invention provides a preparation method of a skin ulcer repairing matrix, which adopts natural extracellular matrix subjected to decellularization, antigen removal and thickening as a biological support, forms cell membranes with multiple layers of cells on the surface of the natural extracellular matrix by composite seed cells, and obtains the skin ulcer repairing matrix after freeze drying treatment, thereby realizing the effects of surface cell stratification of the extracellular matrix and part of cells in the extracellular matrix, effectively protecting and promoting the repair of a wound surface, reducing the formation of scars, having short preparation period, simple process, wide application range and good wound surface repairing effect, being suitable for industrialization, and ensuring that the elasticity and toughness of the healed wound surface are similar to those of normal skin. The freeze-drying sterilization treatment is adopted, so that the expected use effect of the product is ensured, the shelf life can be prolonged, and the requirements on transportation and storage are greatly reduced.

Description

Preparation method of skin ulcer repairing matrix

Technical Field

The invention belongs to the technical field of tissue engineering, and relates to a preparation method of a skin ulcer repairing matrix.

Background

The skin is the first defense barrier of the human body and has the important function of maintaining the steady state of the body, so that the skin deletion injury caused by any reason needs to be treated in time. Small-area superficial skin lesions can be self-repaired by the human body, but the wounds are larger than 20cm in diameter or deep to the dermis and chronic skin ulcer patients need to be treated by other means.

Chronic Skin Ulcer (CSU), also called refractory ulcer, is a common clinical disease. Diabetic foot and venous ulcer are both intractable ulcers. In recent years, with the improvement of living standard and the aging of society, the incidence of Diabetes Mellitus (DM) has been rising year by year. At present, about 9200 thousands of diabetic patients in China can suffer from foot ulcer in 15-25 percent of patients. If left untreated, amputation may result.

For treating various skin defects, the traditional skin grafting method has various defects which cannot be overcome, such as multiple wound surfaces and increased pain of patients; the requirement of large-area implantation cannot be met, and the like. In addition, some intractable ulcer wounds such as diabetic foot and venous ulcer cannot be healed by simple skin grafting. The skin ulcer repair matrix provides a solution to these problems.

Tissue engineered skin is generally prepared from seed cells and scaffold materials. The types of scaffold materials are more, but due to the limitations of the properties of the materials, the treatment method and the like, the currently reported scaffold cannot well meet the cell proliferation requirement or has a complex preparation process. Extracellular matrix (ECM) is a generic term for extracellular substances in connective tissue and is a good scaffold material. It can store, display and release growth factors. There have been many reports demonstrating that extracellular matrix is effective in directing fibroblast growth into and promoting neovascularization. Growth factors secreted by the inoculated cells have important influence on wound healing processes of ulcer, burn, scald and the like. The growth factor can directly promote the proliferation and differentiation of transplanted cells and can also maintain the biological function of the transplanted cells; the reduction of the activity and the relative or absolute lack of the quantity of wound surface local growth factors and receptors thereof such as ulcers, deep burns and scalds and the like are the pathophysiological basis of difficult wound surface healing.

The Chinese patent application No. 200910078357.0 (patent application No. 200910078357.0 is invalid) discloses a method for constructing tissue engineering skin by using human epidermal cells as seed cells and extracellular matrix secreted by human fibroblasts as a biological scaffold. The method has the defects of long culture time (generally 20-50 days), high cost and complex process.

The Chinese patent with patent application number 200810116108.1 (patent right is invalid) mentions a method for constructing tissue engineering skin by taking human fibroblast as seed cell and inoculating the seed cell in gel biomaterial. The method also has the defects of long culture period (generally 15-20 days), high cost, complex process and the like.

The Chinese patent application with the patent application number of 201510100264.9 discloses a tissue engineering skin and application thereof, wherein the tissue engineering skin is constructed by taking Acellular Dermal Matrix (ADM) as a scaffold material and umbilical cord mesenchymal stem cells as seed cells. Although the culture time is shortened, the effect of covering the surface of ADM with cell membranes can be achieved only by coating a layer of gelatin on the surface of the acellular dermal matrix, then performing multiple inoculation, puncturing the surface of ADM and culturing for a period of time. Without gelatin coating, a product with intact cell membranes on the surface could not be obtained. The preparation method does not enable the acellular matrix to fully exert the self-action during cell culture, and is complex in process, high in cost and not suitable for industrialization. Moreover, the preparation method can only form a monolayer cell membrane on the surface of ADM, and growth factors secreted by cells as a protein can be rapidly degraded in vivo, so that the amount of the growth factors secreted by the monolayer cell membrane is far from meeting the effect of regulating the microenvironment of the wound surface, and the repair effect of the growth factors on the wound surface is limited. In addition, almost all the tissue engineering skin products reported at present have the same problems, namely short shelf life and high requirements on storage conditions and transportation due to product forms.

Disclosure of Invention

The invention aims to solve the defects of long culture period, high cost, complex process, unsuitability for industrialization and incapability of meeting the function of regulating the wound microenvironment in the existing tissue engineering skin construction method, so that the repair effect on the wound is limited, the quality guarantee period is short, and the requirements on storage conditions and transportation are high.

The invention provides a preparation method of a skin ulcer repairing matrix, which mainly adopts a natural extracellular matrix subjected to decellularization, antigen removal and thickening as a biological scaffold, forms a cell membrane with a plurality of layers of cells on the surface of the natural extracellular matrix by using composite seed cells, and obtains the skin ulcer repairing matrix after freeze drying treatment;

the seed cells are fibroblasts or stem cells of autologous or allogeneic origin.

The preparation method of the skin ulcer repairing matrix specifically comprises the following steps:

1) step one, preparation of extracellular matrix: soaking the collagen membrane tissue subjected to the cell removal, antigen removal and inactivation treatment with an alkali solution for thickening, then washing with a 0.01-0.05M PBS solution until the pH value is 6-7, soaking with the PBS solution for 1-6 hours to obtain an extracellular matrix, and then irradiating and sterilizing with 10-25 kGy cobalt 60 for later use;

2) step two, preparation of a culture medium:

2.1), preparing a base liquid: mixing DMEM/F12 commercial culture solution and fetal calf serum or newborn calf serum into base solution according to the volume ratio of 9: 1;

2.2), preparing a basic culture medium: adding 1-100 ng of insulin, 1-50 mg of transferrin, 1-30 mug of sodium selenite, 10-600 mug of hydrocortisone, 1-20 mug of epidermal growth factor, 1-100 ng of basic fibroblast growth factor, 1-100 ng of transforming growth factor beta and 1-100 mg of vitamin C according to 500ml of the basic liquid standard to prepare a basic culture medium;

2.3), preparing a culture medium: respectively adding 10-30 mug/ml of bovine pituitary extract, 1-50 mg of adenine, 50-300 ng/ml of HRG and 1-20 ng/ml of platelet derived factor into the basic culture medium to respectively prepare the following four different culture media:

culture medium a: 10-30 mu g/ml of basal medium and bovine pituitary extract;

and (3) culture medium b: a basal medium and adenine of 1-100 mu g/ml;

and (3) a culture medium c: the basic culture medium plus HRG is 50-300 ng/ml;

and (3) a culture medium d: a basal medium and 1-20 ng/ml of platelet-derived factor;

wherein the concentration of the bovine pituitary extract, adenine, HRG and platelet-derived factor is the final concentration;

3) step three, separating and culturing seed cells in vitro: separating seed cells from the required tissue by adopting a tissue adherence method, or separating the seed cells from the required tissue by adopting an enzyme digestion method, or separating the seed cells from the required tissue by adopting a differential adherence method;

and mixing 5-20% fetal bovine serum with F12 or MEM or DMEM or RPMI-1640 culture medium for in vitro subculture at 25-38 deg.C and 3-7% CO₂Concentration;

4) step four, inoculating cells and three-dimensional culture: under sterile environment, 1 × 10⁴～10×10⁶Per cm²Is prepared in the first stepThe extracellular matrix is inoculated with cells, the extracellular matrix is penetrated for 0.2-1 mm for inoculation when the extracellular matrix is inoculated, 0.5-1.0 ml is injected each time, the density of pinholes is 0.1-0.5 per cm²And performing punctate inoculation on the surface of the extracellular matrix, and then uniformly distributing the seed cells on the surface of the extracellular matrix by slight oscillation;

5% CO at 37 ℃₂In an incubator, completely immersing the inoculated extracellular matrix in a culture medium a, culturing for 1-2 days, then replacing a culture medium b, immersing the inoculated extracellular matrix, culturing for 1-2 days, replacing the culture medium c, enabling the liquid level of the culture medium c to be flush with the liquid level of the inoculated extracellular matrix, continuing culturing for 1-2 days, replacing the culture medium d, similarly immersing the inoculated extracellular matrix, and culturing for 1-2 days; changing the culture medium every day during the culture period;

5) step five, freeze-drying, packaging and sterilizing: and (3) freeze-drying the sample cultured in the third step, controlling a freeze-drying program to be reduced to-80 to-20 ℃ at the speed of 5-12 ℃/min, keeping for 1-4 hours, freeze-drying for 10-24 hours, packaging, and performing irradiation sterilization by using 10-25 kGy cobalt 60 to obtain the skin ulcer repairing matrix.

The alkali solution soaking treatment is to soak the collagen membrane tissue for 5 to 30 minutes in 1M alkali solution or 1 to 1 percent sodium hypochlorite for 1 to 2 hours at room temperature, so as to increase the thickness of the collagen membrane tissue by 2 to 7 times.

The alkali solution is any one of sodium hydroxide alkali solution, sodium bicarbonate alkali solution, sodium carbonate alkali solution and potassium hydroxide alkali solution.

The thickness of the collagen membrane tissue is increased by 3 to 5 times.

Uniformly punching holes on the prepared extracellular matrix in the first step, and then irradiating and sterilizing the extracellular matrix by using 10-25 kGy cobalt 60 for later use;

the pore diameter of the pores is 0.5-2 mm, and the pore density of the pores is 0.2-1/cm²And sterilizing for later use.

The collagen membrane tissue is any one of allogenic skin and amniotic membrane;

or any of bovine, porcine-derived skin, pericardium and peritoneum.

The stem cell is any one of umbilical cord mesenchymal stem cell, umbilical cord blood mesenchymal stem cell, skin stem cell, oral mucosa stem cell, dental pulp mesenchymal stem cell, adipose mesenchymal stem cell, bone marrow mesenchymal stem cell, hematopoietic stem cell, liver stem cell and neural stem cell.

Compared with the prior art, the invention has the following advantages:

(1) the method provided by the invention can realize the effect of forming a plurality of layers of cell membranes on the surface of the extracellular matrix by single inoculation, ensure that a plurality of cytokines with sufficient quantity can be secreted when the wound surface is repaired, induce the formation of new vessels and promote the healing of the wound surface. In addition, the effects of protection, support and barrier of the natural extracellular matrix and the effects of guiding cell growth, tissue regeneration and the like of the three-dimensional space structure can effectively protect and promote the repair of the wound surface and reduce the formation of scars.

(2) The collagen membrane after the cell removal treatment is soaked in the alkali liquor, so that the method is simple, and the obtained extracellular matrix collagen fiber structure is looser. The loose collagen fiber structure not only provides a more suitable three-dimensional space structure for the growth and proliferation of cells, but also ensures that nutrient components in the culture medium can be rapidly exchanged and permeated, and greatly promotes the proliferation of the cells. And then washing and soaking by using a PBS solution, so that residual reagents are removed, and the microenvironment for cell growth and proliferation is improved. The formed multilayered cell membrane is more tightly adhered to the extracellular matrix, even the cell part can grow into the matrix, and the acting time of the cell factor is prolonged.

(3) The freeze-drying mode is adopted for processing, so that the expected use effect of the product is ensured, the shelf life can be prolonged, and the requirements on transportation and storage are greatly reduced.

(4) The preparation period is short, the process is simple, the method is suitable for industrialization, the application range is wide, the wound repair effect is good, the scar formation is reduced, and the elasticity and the toughness of the healed wound are similar to those of normal skin.

Drawings

FIG. 1A is a HE picture of bovine pericardium after soaking treatment with lye.

FIG. 1B is a HE picture of bovine pericardium after soaking treatment with lye.

FIG. 2A is a scanning electron micrograph of extracellular matrix surface cell membranes after 2 days of culture.

FIG. 2B is a scanning electron micrograph of extracellular matrix surface cell membranes after 3 days of culture.

FIG. 2C is a scanning electron micrograph of extracellular matrix surface cell membranes after 5 days of culture.

FIG. 3 shows HE staining of extracellular matrix after 5 days of single inoculation of cells.

Fig. 4A is a real object diagram of rat burn and scald wound 1w (1 week) after being treated with the skin ulcer repair matrix.

Fig. 4B is a real image of the control group after treatment of the burn and scald wound of rat 1w (1 week).

Fig. 4C is a real image of the burned and scalded wound surface of rat 3w (3 weeks) after being treated with the skin ulcer repairing matrix.

Fig. 4D is a pictorial representation of the control group after treatment of the burned and scalded wound surface of rat 3w (3 weeks).

Detailed Description

In order to solve the problems of long culture period, high cost, complex process, unsuitability for industrialization and incapability of meeting the function of regulating the wound microenvironment in the existing tissue engineering skin construction method, so that the defects of limited wound repair effect, short quality guarantee period and high requirements on storage conditions and transportation are overcome, the embodiment provides a preparation method of a skin ulcer repair matrix, which comprises the following steps: the seed cells are directly inoculated on the specially treated extracellular matrix, the effects of surface cell stratification of the extracellular matrix and partial cell growth in the extracellular matrix are realized, the freeze-drying sterilization treatment is carried out on the seed cells, the prepared skin ulcer repairing matrix can effectively promote the healing of chronic wounds such as diabetic foot ulcer, venous ulcer, deep burn and scald and the like, the formation of scars can be reduced, and the elasticity and the toughness of the healed wound surface are similar to those of normal skin.

The preparation method of the skin ulcer repairing matrix comprises the steps of taking a natural extracellular matrix subjected to decellularization, antigen removal and thickening as a biological scaffold, forming cell membranes (cells with 2-10 cell membranes) of multiple layers of cells on the surface of the natural extracellular matrix by using composite seed cells, and performing freeze drying treatment to obtain the skin ulcer repairing matrix; wherein the seed cells involved are fibroblasts or embryonic or stem cells of autologous or allogeneic origin.

According to the preparation process, the preparation method of the skin ulcer repair matrix mainly comprises the steps of preparation of extracellular matrix, preparation of culture medium, separation and in-vitro culture of seed cells, cell inoculation, three-dimensional culture, freeze-drying and the like; the preparation process comprises the following steps:

step one, preparation of extracellular matrix:

and (3) cell removal: the collagen membrane tissue used for preparing the extracellular matrix is subjected to decellularization, antigen removal and inactivation treatment after removing fat and other impurities, and specific treatment methods can be any decellularization, antigen removal and inactivation treatment methods described in patent application nos. CN201110145229.0, CN201210251134.1, CN201310358048.5, CN201310117569.1 and CN 201310109216.7. Such as the method mentioned in patent application No. CN 201310109216.7: and (3) soaking the pretreated collagen membrane in a mixed solution of 2.0M potassium chloride and 0.1M ammonia water for 60 minutes, then replacing the soaked collagen membrane, shaking the soaked collagen membrane for 1 hour by using the same amount of purified water, carrying out alternate circulation treatment for 3 times, and finally washing the collagen membrane by using the purified water until the pH value is 6.0-8.0.

Obtaining an extracellular matrix: soaking the treated collagen membrane tissue (collagen membrane for short) in 1M aqueous alkali for 5-30 min, such as sodium hydroxide solution, sodium bicarbonate solution, sodium carbonate solution, potassium hydroxide solution and the like, or soaking in 1 per thousand-1% sodium hypochlorite for 1-2 h, so that the thickness of the collagen membrane is increased by 2-7 times, preferably by 3-5 times. Then washing the cell with a large amount of 0.01-0.05M PBS solution until the pH value is 6-7, and soaking the cell with the PBS solution for 1-6 hours to obtain the extracellular matrix. The step is to make the collagen fiber structure of the collagen membrane more loose through the action of the alkali liquor, as shown in figure 1B (compared with figure 1A, the alkali liquor treatment can effectively loosen the collagen fiber structure), so that the cells can be better adhered and even grow into the collagen fiber structure, and the structure and the performance of the product are more stable.

In addition, the cleaning and soaking by the PBS solution can not only make the microenvironment in the collagen membrane more suitable for the growth and proliferation of cells, but also remove other residual reagents, thereby ensuring that the extracellular matrix does not generate any negative influence on the proliferation of the cells. Cutting the extracellular matrix into a certain specification, such as a circle, a rectangle and the like, or perforating (including perforating and not perforating, and the aperture is 0.5-2 mm) on the extracellular matrix to uniformly distribute pores on the extracellular matrix, wherein the density of the pores is 0.2-1/cm²And performing irradiation sterilization on 10-25 kGy cobalt 60 for later use.

It is obvious that the extracellular matrix is decellularized collagen membrane thickened by alkali liquor, and the main components of the extracellular matrix are type I and type III collagen, including allogeneic skin, amnion and the like, and also including tissue membranes such as skin, pericardium, peritoneum and the like of different sources such as cattle, pigs and the like. Not only has similar components with human skin, but also has certain elasticity and toughness and almost has no immunogenicity. Meanwhile, the treated collagen membrane has a more loose three-dimensional space structure, can guide cells to grow in and tissues to regenerate, enables the healed wound surface to have the characteristic close to normal skin, and can also inhibit scar formation.

Step two: preparation of a culture medium:

mixing DMEM/F12 commercial culture solution and fetal calf serum or newborn calf serum into base solution according to the volume ratio of 9: 1; adding 1-100 ng of insulin, 1-50 mg of transferrin, 1-30 mug of sodium selenite, 10-600 mug of hydrocortisone, 1-20 mug of epidermal cell growth factor, 1-100 ng of basic fibroblast growth factor, 1-100 ng of transforming growth factor beta and 1-100 mg of vitamin C according to a 500ml basic solution standard to prepare a basic culture medium, and then preparing the following culture medium by using the basic culture medium, wherein the concentration of the additives is the final concentration:

culture medium a: 10-30 mu g/ml of basal medium and bovine pituitary extract;

and (3) culture medium b: a basal medium and adenine of 1-100 mu g/ml;

and (3) a culture medium c: the basic culture medium plus HRG is 50-300 ng/ml;

the added bovine pituitary extract, adenine, HRG and platelet derived factor all have the effect of promoting cell growth and proliferation, and are helpful for realizing cell membrane stratification.

Step three, separating and culturing seed cells in vitro: the isolation and in vitro culture of the relevant seed cells can be performed by referring to the prior art, such as the isolation of seed cells from the desired collagen membrane tissue by tissue adherence, or the isolation of seed cells from the desired collagen membrane tissue by enzymatic digestion, or the isolation of seed cells from the desired collagen membrane tissue by differential adherence; and mixing 5-20% fetal bovine serum with F12 or MEM or DMEM or RPMI-1640 culture medium for in vitro subculture at 25-38 deg.C and 3-7% CO₂And (4) concentration.

Step four, inoculating cells and three-dimensional culture: under sterile environment, 1 × 10⁴～10×10⁶Per cm²The seeding density of (a) is to seed the cells on the extracellular matrix. When in inoculation, a sharp-pointed-end device is used for puncturing 0.2-1 mm of extracellular matrix to perform needle-type inoculation, namely injection, wherein 0.5-1.0 ml is injected each time, and the density of pinholes is 0.1-0.5 per cm²And performing punctate inoculation on the extracellular matrix surface, namely dripping the punctate inoculation on the extracellular matrix surface, and then uniformly distributing the cells on the extracellular matrix surface by slight oscillation. The culture conditions are as follows: 37 ℃ and 5% CO₂5% CO at 37 ℃₂In an incubator, completely immersing the inoculated extracellular matrix in a culture medium a, culturing for 1-2 days, then replacing a culture medium b, immersing the inoculated extracellular matrix, culturing for 1-2 days, replacing the culture medium c, enabling the liquid level of the culture medium c to be flush with the liquid level of the inoculated extracellular matrix, continuing culturing for 1-2 days, replacing the culture medium d, similarly immersing the inoculated extracellular matrix, and culturing for 1-2 days; the culture period was changed every day.

The cells mentioned here are seed cells, and are fibroblasts and stem cells of autologous or allogeneic sources, such as umbilical cord mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, skin stem cells, oral mucosa stem cells and dental pulp mesenchymal stem cells, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, hematopoietic stem cells, liver stem cells and neural stem cells, and the like.

The inoculation is preferably carried out on the rough surface of the extracellular matrix, because the collagen fiber structure of the rough surface is looser, and a three-dimensional space can be better provided for the growth and proliferation of cells. And in addition, the structure of collagen fibers (collagen membranes) is loosened by soaking in the alkali liquor in the step one, so that the obtained extracellular matrix has a three-dimensional space more suitable for cell growth and proliferation, and finally, the cells are easy to adhere to the extracellular matrix closely and even are guided to grow into the extracellular matrix. In addition, this seeding method also increases the adhesion of cells to the extracellular matrix and forms a multilayered cell membrane on the surface thereof, as shown in fig. 2A, 2B, 2C (the cell membrane on the surface of the extracellular matrix after fine culture is multilayered) and fig. 3, in which a plurality of layers of cells are closely adhered to the surface of the extracellular matrix after alkali treatment and a certain amount of cells are distributed in the inner superficial layer).

Step five, freeze-drying, packaging and sterilizing: and freeze-drying the cultured sample, controlling the freeze-drying process to reduce the temperature to-80 to-20 ℃ at the speed of 5-12 ℃/min, keeping the temperature for 1-4 hours, and freeze-drying for 10-24 hours to obtain the freeze-dried sample. And packaging the sample, and performing 10-25 kGy cobalt 60 irradiation sterilization again to obtain the skin ulcer repairing matrix.

The skin ulcer repair matrix obtained by the method has the advantages that abundant cytokines in the transplanted product first play a role, the microenvironment for the regeneration of cells and tissues unfavorable to the wound surface is adjusted, and the microcirculation capillary vasospasm is relieved, so that the regeneration repair of the epithelium and the skin vascular remodeling are promoted. At this time, the extracellular matrix mainly functions to protect the wound surface, maintain a moist environment, release cytokines, and the like. After new blood vessels and granulation tissues appear, the extracellular matrix has obvious effect, the natural three-dimensional structure can effectively guide cells to grow in, the wound healing can be protected and promoted, excessive accumulation of formed collagen fibers can be prevented, the formation of scars is reduced, and the elasticity and the toughness of the healed wound are similar to those of normal skin.

The technical scheme of the invention is further explained in detail by combining the examples.

Example 1

After the bovine pericardium is subjected to cell removal treatment and is washed, the bovine pericardium is soaked in 1M sodium hydroxide solution for 5min at room temperature, and then washed by a large amount of 0.01M PBS solution until the pH value is about 7. At the moment, the thickness of the bovine pericardium is increased by 3-4 times. Bovine pericardium was then submerged in PBS solution for 2 h. The bovine pericardium is cut into a 10cm round shape, and is irradiated and sterilized by 10kGy cobalt 60 for standby.

Mixing DMEM/F12 commercial culture solution and 10% fetal calf serum into a base solution according to the volume ratio of 9: 1; then adding 50ng of insulin, 30mg of transferrin, 10 ug of sodium selenite, 60 ug of hydrocortisone, 20 ug of epidermal growth factor, 40ng of basic fibroblast growth factor, 20ng of transforming growth factor beta, 10mg of adenine and 30mg of vitamin C according to the standard of 500ml of basic solution.

Spreading the sterilized bovine pericardium on a sterile culture dish with the diameter of 10cm, and lightly pressing to make the bovine pericardium completely attached to the bottom of the culture dish. Taking umbilical cord mesenchymal stem cells which are subcultured to 2-6 generations as seed cells from umbilical cord of newborn, and culturing at a ratio of 5 × 10⁵/cm²The inoculation density of (2) is to inoculate on the rough surface of bovine pericardium. When inoculating, a 2ml sterile syringe is used to pierce bovine pericardium by 1mm, each injection is 0.5ml, and the density of pinholes is 0.5/cm²The plurality of points are evenly inoculated, then the cells are dripped on the surface of the bovine pericardium and are lightly shaken to be evenly covered on the bovine pericardium. Adding a proper amount of prepared culture medium a to enable the culture medium a to just submerge the bovine pericardium, enabling the bovine pericardium to grow on the wall for 2 hours, then supplementing the culture medium a to enable the liquid level of the culture medium a to be 1cm or so away from the surface of the bovine pericardium, and placing 5% CO at 37 DEG C₂And (3) carrying out shake culture in an incubator for 1 day, then replacing the culture medium b, submerging the bovine pericardium, replacing the culture medium c after 2 days of culture, enabling the liquid level to be flush with the bovine pericardium, replacing the culture medium d after 1 day of continuous culture, similarly submerging the bovine pericardium, and culturing for 2 days. The culture period was changed every day.

And repeatedly washing the cultured bovine pericardium by using sterile PBS solution, then controlling the freeze-drying program to reduce the temperature to-80 ℃ at the speed of 5 ℃/min, keeping the temperature for 1 hour, and freeze-drying for 10 hours. Freeze-drying, packaging, and performing 10kGy cobalt 60 irradiation sterilization again to obtain the skin ulcer repairing matrix.

Example 2

After the bovine pericardium is subjected to cell removal treatment and is cleaned, the bovine pericardium is soaked in 1 per mill of sodium hypochlorite solution for 2 hours at room temperature, and then is cleaned by a large amount of 0.05M PBS solution until the pH value is about 7. At the moment, the thickness of the bovine pericardium is increased by 3-6 times. Bovine pericardium was then submerged in PBS solution for 6 h. The bovine pericardium is cut into a 10cm circle, and is irradiated and sterilized by 25kGy cobalt 60 for standby.

Mixing DMEM/F12 commercial culture solution and 10% fetal calf serum into a base solution according to the volume ratio of 9: 1; then according to the standard of 500ml of basic liquid, 60ng of insulin, 50mg of transferrin, 20 ug of sodium selenite, 100 ug of hydrocortisone, 20 ug of epidermal growth factor, 50ng of basic fibroblast growth factor, 40ng of transforming growth factor beta, 5mg of bovine pituitary extract and 50mg of vitamin C are added.

Spreading the sterilized bovine pericardium on a sterile culture dish with the diameter of 10cm, and lightly pressing to make the bovine pericardium completely attached to the bottom of the culture dish. Using epidermal stem cells obtained by trypsinizing human skin, separating and in vitro culturing as seed cells, and culturing at a ratio of 1 × 10⁴/cm²The inoculation density of (2) is to inoculate on the rough surface of bovine pericardium. When inoculating, a 2ml sterile syringe is used to pierce bovine pericardium by 0.5mm, each injection is 0.6ml, and the density of pinholes is 0.3/cm²The plurality of points are evenly inoculated, then the cells are dripped on the surface of the bovine pericardium and are lightly shaken to be evenly covered on the bovine pericardium. Adding a proper amount of prepared culture medium a to enable the culture medium a to just submerge the bovine pericardium and grow for 2 hours in an adherent manner. Then the culture medium a is supplemented to ensure that the liquid level of the culture medium a is about 1cm away from the surface of the bovine pericardium. Put in 5% CO at 37 DEG C₂And carrying out shaking culture in an incubator. Culturing for 2 days, then replacing the culture medium b, submerging the bovine pericardium, replacing the culture medium c after culturing for 1 day, keeping the liquid level at the same level with the bovine pericardium, continuing culturing for 2 days, replacing the culture medium d, submerging the bovine pericardium similarly, and culturing for 2 days. The culture period was changed every day.

And repeatedly washing the cultured bovine pericardium by using sterile PBS solution, then controlling the freeze-drying program to reduce the temperature to-20 ℃ at the speed of 12 ℃/min, keeping the temperature for 4 hours, and freeze-drying for 24 hours. Freeze-drying, packaging, and performing 25kGy cobalt 60 irradiation sterilization again to obtain the skin ulcer repair matrix.

Example 3

After the pig peritoneal membrane is subjected to cell removal treatment and washed, the pig peritoneal membrane is soaked in a 1% sodium hypochlorite solution for 1h at room temperature, and then washed by a large amount of 0.05M PBS solution until the pH value is about 7. The thickness of the pig abdominal membrane is increased by 4-7 times. The porcine peritoneal membrane was then immersed in the PBS solution for 6 h. Cutting pig abdominal membrane into 10cm circle, and sterilizing with 10kGy Co 60 irradiation.

Mixing DMEM/F12 commercial culture solution and 10% newborn calf serum into a base solution according to the volume ratio of 9: 1; then 10ng of insulin, 1mg of transferrin, 1 ug of sodium selenite, 600 ug of hydrocortisone, 1 ug of epidermal growth factor, 100ng of basic fibroblast growth factor, 100ng of transforming growth factor beta, HRG15mg and vitamin C100mg are added according to the standard of 500ml of basic liquid.

And (3) flatly paving the sterilized porcine peritoneal membrane on a sterile culture dish with the diameter of 10cm, and lightly pressing the porcine peritoneal membrane to be completely attached to the bottom of the culture dish. Culturing cord blood stem cell in vitro as seed cell at 10 × 10⁷/cm²The inoculation density of (2) is to inoculate on the rough surface of the porcine peritoneal membrane. When inoculating, a 2ml sterile syringe is used to pierce bovine pericardium by 0.2mm, each injection is 1ml, and the density of pinholes is 0.4/cm²The multiple spots are inoculated uniformly, then the cells are dripped on the surface of the porcine peritoneal membrane, and the cells are gently shaken to be uniformly covered on the porcine peritoneal membrane. Adding a proper amount of prepared culture medium a to ensure that the pig just submerges the peritoneum of the pig and grows for 2 hours in an adherent manner. Then the culture medium a is supplemented to ensure that the liquid level is about 1cm away from the surface of the porcine peritoneal membrane, and 5 percent CO is added at the temperature of 37 DEG C₂Carrying out shaking culture in an incubator for 2 days, then replacing the culture medium b, submerging the peritoneum of the pig, replacing the culture medium c after 1 day of culture, enabling the liquid level to be flush with the peritoneum of the pig, replacing the culture medium d after 2 days of continuous culture, similarly submerging the peritoneum of the pig, and culturing for 1 day. Changing the culture medium every day during the culture period;

repeatedly washing the cultured porcine peritoneal membrane with sterile PBS solution, controlling the freeze-drying process to reduce the temperature to-40 ℃ at the speed of 8 ℃/min, keeping the temperature for 3 hours, and freeze-drying for 16 hours. Freeze-drying, packaging, and performing 25kGy cobalt 60 irradiation sterilization again to obtain the skin ulcer repair matrix.

Example 4

After the pig dermis is subjected to cell removal treatment and washing, the pig dermis is soaked in 1M potassium hydroxide solution for 1 hour at room temperature, and then washed by a large amount of 0.05M PBS solution until the pH value is about 7. At the moment, the thickness of the pig dermis is increased by 2-4 times. The pig dermis was then submerged in the PBS solution for 4 hours. Cutting pig dermis into 10cm circle, and sterilizing with 15kGy cobalt 60 irradiation for use.

Mixing DMEM commercial culture solution and 10% newborn calf serum into base solution according to the volume ratio of 9: 1; then adding 1ng of insulin, 10mg of transferrin, 5 ug of sodium selenite, 80 ug of hydrocortisone, 15 ug of epidermal growth factor, 30ng of basic fibroblast growth factor, 70ng of transforming growth factor beta, 5mg of platelet derived factor and vitamin C80mg according to 500ml of basic liquid standard.

And (3) spreading the sterilized pig dermis on a sterile culture dish with the diameter of 10cm, and lightly pressing to enable the sterilized pig dermis to be completely attached to the bottom of the culture dish. Taking fibroblast obtained by carrying out enzyme digestion treatment and separation in vitro culture on neonatal foreskin as seed cell according to the proportion of 5 multiplied by 10⁶/cm²The inoculation density of (2) is to inoculate on the rough surface of the porcine peritoneal membrane. When inoculating, the pig dermis is first punctured with 2ml sterile syringe with 0.3mm, each injection is 0.1ml, and the density of needle holes is 0.1/cm²The cells were then dropped onto the surface of the pig dermis and gently shaken to cover the pig dermis uniformly. Adding a proper amount of prepared culture medium a to enable the culture medium a to just submerge the pig dermis, enabling the pig dermis to grow for 2 hours in an adherent manner, and then supplementing the culture medium a to enable the liquid level of the culture medium a to be about 1cm away from the surface of the pig dermis. Put in 5% CO at 37 DEG C₂And (3) carrying out shake culture in an incubator for 1 day, then replacing the culture medium b to submerge the pig dermis, replacing the culture medium c after 1 day of culture, enabling the liquid level to be flush with the pig dermis, replacing the culture medium d after 2 days of continuous culture, similarly submerging the pig dermis, and culturing for 1 day. Changing the culture medium every day during the culture period;

and repeatedly washing the cultured pig dermis by using sterile PBS solution, then controlling a freeze-drying program to reduce the temperature to-60 ℃ at the speed of 6 ℃/min, keeping the temperature for 3 hours, and freeze-drying the pig dermis for 20 hours. Freeze-drying, packaging, and performing 15kGy cobalt 60 irradiation sterilization again to obtain the skin ulcer repairing matrix.

Example 5

10 SD rats with weight of 200-250 g and half male and female are bred in cages for 3d (short for "day" in English day), fasted one day before scald, and are injected with pentobarbital sodium (35mg/kg) in the abdominal cavity for anesthesia, shaved on the back and wiped to dry the skin. A circular wound surface with the diameter of 2cm is manufactured on two sides of the back of a rat by using a self-control handheld scald apparatus with the spine as a midline, the scald temperature is 80 ℃, and the scald time is 5 s. Simultaneously, 10mL of ringer's solution is injected into the abdominal cavity to resist shock, and the feed is raised in cages. The histopathological examination shows that the scald degree is deep II. And (3) removing necrotic skin tissues after scalding for 3d, treating the wound surface, cutting the rehydrated skin ulcer repair matrix (material group) and the bovine pericardium (control group) without inoculated cells into proper sizes, respectively covering the wound surface with the rehydrated skin ulcer repair matrix (material group) and the bovine pericardium without inoculated cells, suturing, packaging and fixing. The injection is made by injecting 0.2mL of normal saline (20000IU/mL) containing penicillin into abdominal cavity to prevent infection, and the rats are raised in cages.

The results of photographing after 1w (material group, FIG. 4A; control group, FIG. 4B, w is abbreviation of Week in Weeks of the week) and 3w (material group, FIG. 4C; control group, FIG. 4D) show:

the material group can be better fused with the autologous skin of a rat 1w after transplantation, the number of capillary vessels in the transplant is gradually increased along with the time, and the graft basically heals after 3w with less scars. The control group did not heal completely and the graft area was scarred and darker in color. The results show that the material group can promote the healing of the wound surface and reduce the scar formation in the healing process of the burn/scald wound surface.

And observing the healing time of the wound surface, the healing rate of the wound surface, the inflammatory reaction of the wound surface, the existence of anaphylactic reaction and the like after the application of the material combination control group. The calculation time points of the wound healing rate are selected from 11 days, 18 days and 25 days after operation, and the calculation formula is as follows:

the fixed-time wound healing rate is (area of wound before treatment-area of wound at fixed time)/area of wound before treatment × 100%.

In the observation process, the material group had a small inflammatory reaction in the early stage, no anaphylaxis occurred, and the results of the wound healing time and the healing rate are shown in table 1. The mean healing time of the wound surface after the application of the material group was 18 days, and that of the control group was 25 days. The wound repair effect of the application material group is obviously better than that of the control group.

TABLE 1 healing time and rate of deep II degree burn/scald wound

Claims

1. A preparation method of a skin ulcer repairing matrix is characterized by comprising the following steps: taking the natural extracellular matrix subjected to decellularization, antigen removal and thickening as a biological scaffold, forming a cell membrane of a plurality of layers of cells with 2-10 layers on the surface of the natural extracellular matrix by using the composite seed cells, and performing freeze drying treatment to obtain the skin ulcer repairing matrix;

the seed cells are fibroblasts or stem cells of autologous or allogeneic sources;

the preparation method of the skin ulcer repairing matrix comprises the following steps:

2) step two, preparation of a culture medium:

culture medium a: 10-30 mu g/ml of basal medium and bovine pituitary extract;

and (3) culture medium b: a basal medium and adenine of 1-100 mu g/ml;

and (3) a culture medium c: the basic culture medium plus HRG is 50-300 ng/ml;

3) step three, separating and culturing seed cells in vitro: separating seed cells from the required tissue by adopting a tissue adherence method, or separating seed cells from the required tissue by adopting an enzyme digestion method, or separating seed cells from the required tissue by adopting a differential adherence method;

and mixing 5-20% fetal calf serum with a F12 or MEM or DMEM or RPMI-1640 culture medium for in-vitro subculture, wherein the culture temperature is 25-38 ℃, and the concentration of CO2 is 3-7%;

4) step four, inoculating cells and three-dimensional culture: under the aseptic environment, the number of the cells is 1 multiplied by 104 to 10 multiplied by 106 per cm²The inoculation density of (A) is to inoculate cells on the extracellular matrix prepared in the step one, the cells are penetrated into the extracellular matrix for 0.2-1 mm to inoculate during inoculation, 0.1-1.0 ml is injected every time, and the density of pinholes is 0.1-0.5 per cm²And performing punctate inoculation on the surface of the extracellular matrix, and then uniformly distributing the seed cells on the surface of the extracellular matrix by slight oscillation;

5% CO at 37 ℃₂In the incubator, the inoculated extracellular matrix is completely immersed in the culture medium a and cultured for 1-2 days,replacing a culture medium b, immersing the inoculated extracellular matrix, culturing for 1-2 days, replacing with a culture medium c, enabling the liquid level of the culture medium c to be flush with the inoculated extracellular matrix, continuously culturing for 1-2 days, replacing with a culture medium d, immersing the inoculated extracellular matrix for 1-2 days; changing the culture medium every day during the culture period;

2. The method for preparing a skin ulcer healing matrix according to claim 1, wherein: the alkali solution soaking treatment is to soak the collagen membrane tissue for 5 to 30 minutes by using 1M alkali solution or for 1 to 2 hours by using 1 per thousand to 1 percent sodium hypochlorite at room temperature, and then the thickness of the collagen membrane tissue is increased by 2 to 7 times.

3. The method for preparing a skin ulcer healing matrix according to claim 2, wherein: the alkali solution is any one of sodium hydroxide alkali solution, sodium bicarbonate alkali solution, sodium carbonate alkali solution and potassium hydroxide alkali solution.

4. The method for preparing a skin ulcer healing matrix according to claim 2, wherein: the thickness of the collagen membrane tissue is increased by 3-5 times.

5. The method for preparing a matrix for skin ulcer repair according to claim 1, wherein: uniformly punching holes on the prepared extracellular matrix in the step one, and then irradiating and sterilizing the extracellular matrix by using 10-25 kGy cobalt 60 for later use;

the aperture of the holes is 0.5-2 mm, the hole density of the holes is 0.2-1/cm 2, and the holes are sterilized for later use.

6. The method for preparing a matrix for skin ulcer repair according to claim 1, wherein: the collagen membrane tissue is any one of allogeneic skin and amniotic membrane;

or any of bovine, porcine-derived skin, pericardium and peritoneum.

7. The method for preparing a matrix for skin ulcer repair according to any one of claims 1 to 6, wherein: the stem cell is any one of umbilical cord mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, skin stem cells, oral mucosa stem cells, dental pulp mesenchymal stem cells, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, hematopoietic stem cells, liver stem cells and neural stem cells.