CN112245653A

CN112245653A - Protein film dressing assisting wound healing based on cooperation of three bioactive scaffold materials and cell trophic factors

Info

Publication number: CN112245653A
Application number: CN202011264888.1A
Authority: CN
Inventors: 朱国君; 罗晓丹
Original assignee: Hangzhou Yiseqi Biotechnology Co ltd
Current assignee: Hangzhou Yiseqi Biotechnology Co ltd
Priority date: 2020-11-13
Filing date: 2020-11-13
Publication date: 2021-01-22
Anticipated expiration: 2040-11-13
Also published as: CN112245653B

Abstract

The invention provides a protein film dressing assisting wound healing based on cooperation of three bioactive scaffold materials and cell trophic factors, which comprises the following components: keratin, oxidized keratin, schizophyllan powder, culture supernatant of pluripotent stem cells, chlorella extract, glycerol glucoside, chitosan powder, filtrate of fermentation products of the leuconostoc/radish roots, glycerol and water. The protein film dressing provided by the invention is rich in physiological structural proteins and biological polysaccharides which are important in functionality, and various nutritional factors and growth factor groups required by cells, is closer to the protein source of a human body, and does not generate immunological rejection; is helpful for rapid recovery of acute wound, and can rapidly coagulate blood, stop exudation, and resist inflammation; for chronic wound surface, it can convert from inflammation stage to repair stage, and prevent skin oxidation and pigmentation caused by free radicals.

Description

Protein film dressing assisting wound healing based on cooperation of three bioactive scaffold materials and cell trophic factors

Technical Field

The invention relates to the field of biological products, in particular to a protein film dressing assisting wound healing based on three bioactive scaffold materials and a cell trophic factor and application of the protein film dressing in the medical field.

Background

The deep understanding of the pathophysiological mechanisms of wound healing can improve the level of wound care, and the exploration and optimization of materials hopes to bring a more efficient wound care scheme. Wound healing is one of the most complex processes in multicellular organisms, involving numerous signaling within and between various cells. Physical, chemical and cytokine involvement in biological processes are the major targets for wound repair. The healing mechanisms of skin wounds can be divided into repair and regeneration, with clear and important differences between the two types of healing. The characteristics and properties of the new tissue in wound repair are far inferior to the original tissue, and the goal of wound regeneration is to reconstruct the damaged tissue with the same tissue as the original tissue to restore the shape and function of the damaged tissue. Wounds with different properties and sizes have certain regularity and wide universality in the process of tissue integrity repair, the cellular immune response is started from wound hemostasis to an organism, the invasion cells and the necrotic tissues are eliminated through inflammatory reaction, then the debridement stage is completed, the regeneration of the granulation tissues and the epithelial cell crawling is overlapped in the debridement later stage, and finally the reconstruction and the repair of the tissues are completed. Normal processes include coagulation, inflammatory reactions, angiogenesis, matrix deposition, wound contraction, epithelialization. There are a variety of growth factors, cytokines, integrins, keratins, matrix metalloproteinases, chemokines, and extracellular macromolecules involved in and regulate the complex wound healing process. Growth factors play an essential role in wound healing and tissue regeneration. Signaling proteins affect the metabolism of other cells. Each growth factor has more than one effect on the wound healing process, which acts by binding to specific receptors on the surface of the target cell membrane. These effects include promoting chemotaxis, stimulating cell migration and proliferation, stimulating cell up-regulation of protein production. These growth factors not only regulate cell migration and proliferation, but also reconstitute extracellular matrix and promote angiogenesis, creating an ideal environment for skin wound healing.

After damage to the skin, platelets are stimulated to aggregate at the damaged site, forming a fibrin clot, promoting hemostasis and attracting several different cells to the wound. Secretion of growth factors and cytokines, as well as other coagulation factors required for the coagulation cascade, also occurs in the first phase of wound healing. Coagulation and platelet degranulation activate the inflammatory response phase. The main feature of this phase is the release of serotonin, histamine and bioactive factors, resulting in increased capillary permeability, which in turn attracts inflammatory cells to the wound area, including neutrophils, leukocytes and macrophages. The highest concentration of neutrophils present at the wound site, 1-2 days after injury, plays a major role in preventing bacterial infection with the help of macrophages, they also activate keratinocytes, fibroblasts and immune cells. At the end of the inflammatory phase of wound healing, macrophages develop an anti-inflammatory, profibrotic phenotype, secreting TGF- β, interleukins, and tumor necrosis factor. These growth factors stimulate the onset of the proliferative phase, i.e., fibrous tissue proliferation, matrix precipitation, angiogenesis, re-epithelialization. During the proliferative phase, fibroblasts are recruited from the surrounding intact tissue, and VEGF stimulates the migration of endothelial cells and promotes the formation of new blood vessels. Activated fibroblasts are responsible for the secretion of extracellular matrix, which contains high levels of immature type III collagen, in contrast to type I collagen found in normal skin and mature scars. The highest concentration of collagen in the wound occurs approximately three weeks after the initial injury. At the same time, starting 3-5 days after the injury, fibrin emboli begin to be replaced by granulation tissue and the wound begins to contract. Eventually, the final phase of wound healing, known as the remodeling or maturation phase, occurs, when a reduction in cellular components within the wound area occurs due to migration and apoptosis. Remodeling is a dynamic process during which tight cross-links can form between collagen and other protein molecules, increasing the tensile strength of mature scars, up to 80% of intact skin. Furthermore, as the immature type III collagen is converted to mature type I collagen, the ratio of type III and type I collagen decreases, a process that can last for up to 2 years.

The wound dressing has high requirements on the biocompatibility and the bioactivity of wound repair materials, the number of suitable materials is small, many protein film dressings in the market are single in source, the technical purification capability at that time is affected, the material selection purity is low, the effect cannot be ensured if an effective amino acid signal sequence cannot be obtained, the process is immature, and the large-scale production cannot be realized. Some dressings in the market are even added with crude Chinese herbal medicines, allergic inflammation is easily caused in the early stage, the inflammatory period of wounds is prolonged, the later-stage healing quality of the wounds is poor, and the wounds are easily prone to become deep. An ideal wound dressing should satisfy the following conditions: excessive seepage and toxin can be removed; high wettability at the wound-dressing interface, allowing gas exchange; providing thermal isolation to prevent secondary infection; no particles and toxic components; can not cause wound when being replaced.

After the body tissue is injured, cells crawl onto collagen scaffolds in the wound, as they climb on spider webs, and as they move, activate and chemotactic active repair proteins to begin repairing the injured tissue. The biological scaffold material can attract stem cells and fibroblasts of an injured person to enter the biological scaffold material, provides places for attaching, growing and proliferating the new cells, and can improve the division and proliferation capacity of keratinocytes and fibroblasts, so that an effective amino acid sequence is needed, and the biological scaffold material can be used as a biological recognition signal to promote cell adhesion and form new tissues. Whether the microstructure of the support material can simulate the three-dimensional structure in the natural state to the maximum extent has important influence, the micropores are breathable, have certain toughness to prevent wound contraction and fracture, and the integrity of the support structure is ensured. Good biocompatibility and unique mechanical properties are required at the same time, and biological material mixing can improve the biological properties of each other, complement the advantages and contribute to accelerating wound healing. An ideal stent would have the following advantages: 1. the wound surface is adhered, and the loss of body fluid and water is prevented; 2. the wound dressing is breathable and moisture-permeable, and can absorb seepage and improve the microenvironment of a wound surface; 3. providing a biological barrier to prevent wound infection and harmful bacteria invasion; 4. the degradation time is consistent with the healing or regeneration time of the wound surface; 5. no inflammatory reaction, toxic reaction and immunological rejection reaction; 6. function and morphology are maintained during the healing period of the tissue.

Disclosure of Invention

Therefore, in order to solve the above problems, the invention provides a protein film dressing for assisting wound healing based on three bioactive scaffold materials and a cell trophic factor, which is realized by the following technical scheme:

a protein film dressing assisting wound healing based on three bioactive scaffold materials and a cell trophic factor comprises the following components in percentage by mass: 15-50% of keratin, 10-50% of oxidized keratin, 0.05-1% of schizophyllan powder, 1-10% of pluripotent stem cell conditioned culture supernatant, 1-25% of chlorella extract, 1-10% of glycerol glucoside, 0.05-1% of chitosan powder, 1-5% of a Leuconostoc/radish root fermentation product filtrate, 2-5% of glycerol and the balance of water.

Further, the keratin is preferably 15-40%, the oxidized keratin is preferably 10-40%, the schizophyllan powder is preferably 0.05-0.8%, and the culture supernatant of the pluripotent stem cell condition is preferably 1-8%.

Further, the keratin is preferably 20-40%, the oxidized keratin is preferably 15-40%, the schizophyllan powder is 0.1-0.8%, and the culture supernatant of the pluripotent stem cell condition is preferably 2-8%.

Further, the keratin is preferably 25-40%, the oxidized keratin is preferably 20-40%, the schizophyllan powder is preferably 0.2-0.8%, and the culture supernatant of the pluripotent stem cell condition is preferably 4-8%.

Further, the keratin is preferably 25-35%, the oxidized keratin is preferably 20-30%, the schizophyllan powder is preferably 0.2-0.6%, and the culture supernatant of the pluripotent stem cell condition is preferably 4-6%.

Further, the protein film dressing can be applied to the medical field, particularly to wound repair, and the protein film dressing is directly applied to an affected part or is prepared into a wound repair patch and the like to cover a wound so as to promote wound healing.

The three bioactive scaffold materials in the protein film dressing provided by the invention are keratin, oxidized keratin and schizophyllan powder respectively. Wherein the keratin has microporous double-helix structure alpha-keratin and is derived from wool and/or silk; the oxidized keratin is derived from wool; schizophyllan powder is a bioactive polysaccharide with a three-helix three-dimensional structure. The keratin and the oxidized keratin are macromolecular keratin scaffold materials with two different conformation double helix structures, the specific average molecular weight is about 40000, the keratin and the oxidized keratin and a protein film formed by schizophyllan have good combination characteristics, and a continuous film with combination force can be formed on a wound to form a stable protective film; meanwhile, the active cystine with high concentration can eliminate free radicals. The active protein can promote the division and proliferation capacity of high keratinocyte and fibroblast, and the alpha-keratin contains RGD signal sequence similar to fibronectin, can be specifically combined with integrin on cell membrane, and effectively promotes the adhesion of cell to biological material.

In addition, the bioactive scaffold material is also beneficial to the aggregative transmission of the culture supernatant of the pluripotent stem cell condition and chlorella extract, the centralized repair, the acceleration of wound healing and the reconstruction of tissues. In skin basic function, nutrients are important for wound healing, and microscopically nutrients are essential for good cell proliferation, cell overall function and cell communication. Skin cells, like other human cells, are heterotrophic: for proper functioning, the cells take up nutrients, water and mineral salts from the extracellular matrix, and if the skin fails to absorb the appropriate nutrients, the quality of wound healing must not be as good. Chlorella is an alga that is believed to contain the most antioxidants, and the main matrix is protein, in addition to which carbohydrates, minerals, fatty acids, chlorophyll and other nutrients are contained, thus playing an important role in cell repair.

In the process of reparation, a large amount of energy is consumed, the power of cells is very necessary to be maintained, the synthesis of ATP can be improved by 129% through the glycerol glucoside, the glycerol glucoside is the most main active substance for the survival and reactivation of the cells in extreme environments, the osmotic balance of the cells is maintained, the cells adapt to the environment quickly, the hydration is sufficient, the water distribution is improved, the water is locked firmly, the lasting moisture retention is ensured, the activity of the cells is improved, the energy is supplied, the mitochondria are protected, and the protein oxidation is resisted.

The culture supernatant of pluripotent stem cells is processed by a special process, is generated by new cells in a simulated embryo environment, contains a mixture of different cell signal transduction raw materials and has complex components. Growth factors known to be involved in the wound healing process include platelet derived growth factor, epidermal growth factor, fibroblast growth factor, insulin-like growth factor, vascular endothelial growth factor, transforming growth factor, keratinocyte growth factor; growth factors are a group of water-soluble and diffusible polypeptide substances that regulate the growth, differentiation, proliferation and cellular metabolism of various cells. The pluripotent stem cell conditioned culture supernatant contains keratinocyte growth factor, soluble human extracellular matrix protein such as collagen, and key proteins for supporting epidermal stem cells and helping to regenerate skin; can provide the structure and signal required by cell and tissue growth, has anti-inflammatory property, and can stimulate the cell growth of damaged area to promote regeneration.

In summary, the above embodiments of the present application may have one or more of the following advantages or benefits:

1. the biological protein is rich in functionally important physiological structural proteins and biological polysaccharides and various nutritional factors and growth factor groups required by cells, is closer to the protein source of a human body, and does not generate immunological rejection;

2. contains whole protein macromolecules and micromolecular polypeptides, can form a sacrificial protein protective film, and protects the skin from being damaged;

3. the framework support structure with biological activity stimulates the repair of surrounding tissues, the action mechanism is similar to that of a zipper, and amino acid pairing combination is an important biological material for cell growth and protein synthesis;

4. the composition is beneficial to matrix deposition, the epithelial repair is faster, and the composition meets the physiological process of normal skin growth and reduces the generation of scars;

5. is helpful for rapid recovery of acute wound, and can rapidly coagulate blood, stop exudation, and resist inflammation;

6. for chronic wound surface, it can convert from inflammation stage to repair stage, and prevent skin oxidation and pigmentation caused by free radicals.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.

FIG. 1 is a comparative graph of wound repair in patient 1;

FIG. 2 is a comparative graph of wound repair for patient 2;

FIG. 3 is a comparative graph of wound repair of patient 3;

fig. 4 is a comparative graph of wound repair for patient 4.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

[ example 1 ]

A protein film dressing assisting wound healing based on three bioactive scaffold materials and a cell trophic factor comprises the following components in percentage by mass: 15% of keratin, 50% of oxidized keratin, 0.05% of schizophyllan powder, 10% of culture supernatant of pluripotent stem cell conditions, 1% of chlorella extract, 5% of glycerol glucoside, 0.05% of chitosan deacetylate powder, 1% of tricholoma minuense/radish root fermentation product filtrate, 2% of glycerol and 15.9% of water.

Patient 1 had a medical history of over 20 years, had no healing of the wound on the arm, had red and swollen affected parts, damaged epidermis, and occasional weeping; after one month of treatment, the effect of the protein film dressing prepared in this example is shown in fig. 1, and it can be seen that the wound has scabbed.

[ example 2 ]

A protein film dressing assisting wound healing based on three bioactive scaffold materials and a cell trophic factor comprises the following components in percentage by mass: keratin 50%, oxidized keratin 10%, schizophyllan powder 1%, pluripotent stem cell condition culture supernatant 6%, chlorella extract 5%, glycerol glucoside 10%, chitosan powder 0.2%, staphylococcus aureus/radish root fermentation product filtrate 5%, glycerol 3% and water 9.8%.

Many skin lesions on the back of patient 2 appeared in a red and swollen state, sometimes accompanied by itching according to dictation, and had a history of 20 years, the wound never healed; after one month of treatment, the effect of the protein film dressing prepared by the embodiment is shown in figure 2, the wound is completely healed and slightly reddened, and the skin can be automatically repaired.

[ example 3 ]

A protein film dressing assisting wound healing based on three bioactive scaffold materials and a cell trophic factor comprises the following components in percentage by mass: 20% of keratin, 30% of oxidized keratin, 0.2% of schizophyllan powder, 1% of culture supernatant of pluripotent stem cells, 25% of chlorella extract, 2% of glycerol glucoside, 0.8% of chitosan deacetylate powder, 3% of tricholoma minuense/radish root fermentation product filtrate, 4% of glycerol and 14% of water.

The wound on the hand of patient 3 is a chronic wound and does not heal for 3 years; the effect of the protein film dressing prepared in the example after 2 days is shown in fig. 3, and it is obvious that the skin becomes reddish after 2 days of treatment, but the skin is no longer red and swollen, and the wound is healed.

[ example 4 ]

A protein film dressing assisting wound healing based on three bioactive scaffold materials and a cell trophic factor comprises the following components in percentage by mass: 40% of keratin, 20% of oxidized keratin, 0.8% of schizophyllan powder, 3% of culture supernatant of pluripotent stem cells, 15% of chlorella extract, 1% of glycerol glucoside, 1% of chitosan deacetylate powder, 2% of tricholoma minuense/radish root fermentation product filtrate, 5% of glycerol and 12.2% of water.

The feet of the patient 4 have seasonal herpes, have a history of 5 years, are not cured radically all the time, have outbreaks sometimes, have skin chapping and peeling during the onset of diseases, and have pruritus at the herpes zoster position; after one week of treatment with the protein film dressing prepared in this example, the effect is shown in fig. 4, the skin color of the affected part is slightly darker than elsewhere, but the herpes has completely healed, and the skin is no longer dry and skinned.

The invention provides a protein film dressing assisting wound healing based on three bioactive scaffold materials and a cell trophic factor, which can also comprise the following components in percentage by mass:

finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.

Claims

1. A protein film dressing assisting wound healing based on three bioactive scaffold materials and a cell trophic factor is characterized by comprising the following components in percentage by mass: 15-50% of keratin, 10-50% of oxidized keratin, 0.05-1% of schizophyllan powder, 1-10% of pluripotent stem cell conditioned culture supernatant, 1-25% of chlorella extract, 1-10% of glycerol glucoside, 0.05-1% of chitosan powder, 1-5% of a Leuconostoc/radish root fermentation product filtrate, 2-5% of glycerol and the balance of water.

2. The protein film dressing according to claim 1, wherein the keratin is preferably 15-40%, the oxidized keratin is preferably 10-40%, the schizophyllan powder is preferably 0.05-0.8%, and the pluripotent stem cell conditioned culture supernatant is preferably 1-8%.

3. The protein film dressing according to claim 1, wherein the keratin is preferably 20-40%, the oxidized keratin is preferably 15-40%, the schizophyllan powder is 0.1-0.8%, and the pluripotent stem cell conditioned culture supernatant is preferably 2-8%.

4. The protein film dressing according to claim 1, wherein the keratin is preferably 25-40%, the oxidized keratin is preferably 20-40%, the schizophyllan powder is preferably 0.2-0.8%, and the pluripotent stem cell conditioned culture supernatant is preferably 4-8%.

5. The protein film dressing according to claim 1, wherein the keratin is preferably 25-35%, the oxidized keratin is preferably 20-30%, the schizophyllan powder is preferably 0.2-0.6%, and the pluripotent stem cell conditioned culture supernatant is preferably 4-6%.

6. A protein film dressing according to any of claims 1 to 5, wherein the keratin is derived from wool and/or silk.

7. A protein film dressing according to any one of claims 1 to 5, wherein the oxidised keratin protein is derived from wool.

8. The protein film dressing of any one of claims 1-5, wherein the pluripotent stem cell conditioned culture supernatant is produced from novacells in a simulated embryonic environment.

9. Use of a protein film dressing according to any one of claims 1 to 8 in the medical field.