CN1319605C - Collagen-chitin and silicon rubber bilayer skin regeneration support and its preparation method - Google Patents

Abstract

The invention discloses a collagen-chitosan and silicon rubber double-layer skin regeneration support and a preparation method thereof. The skin regeneration scaffold is formed by bonding a collagen/chitosan porous scaffold and a silicon rubber film through an adhesive with good biocompatibility. Among them, the collagen/chitosan porous scaffold can effectively induce the migration, proliferation and differentiation of cells in the defect tissue, and induce the regeneration of the defect dermal tissue in situ; the silicone rubber film plays the role of a temporary epidermis, which can effectively control the volatilization and Prevent the invasion of bacteria, etc., and play a temporary protective role on the wound surface. The collagen-chitosan/silicone rubber composite scaffold prepared by the invention has good biocompatibility, controllable degradation rate and excellent wound repair ability, and has good clinical application prospect.

Description

Collagen-chitosan and silicone rubber double-layer skin regeneration scaffold and its preparation method

technical field

The invention relates to a double-layer skin regeneration support and a preparation method thereof. Specifically, it relates to a collagen-chitosan and silicon rubber double-layer skin regeneration support and a preparation method thereof.

Background technique

Burns or ulcers of the skin are very common surgical diseases, such as wounds in various accidents and wars, and skin ulcers caused by self-diseases such as diabetes. Skin defects can affect the patient's appearance in mild cases, and in severe cases can cause infection or a large loss of electrolytes and water, leading to the death of the patient. Although skin tissue has a strong regenerative ability, for a large area of full-thickness skin defect, the skin loses its ability to spontaneously regenerate. The key to the treatment of full-thickness skin defects is to seal the wound as early as possible, so as to reduce the consumption of the body, the disturbance of the internal environment, and the occurrence of complications such as microbial invasion. So far, autologous skin grafting is still the most effective method for clinical treatment of full-thickness skin defects. However, this method faces the shortcoming of insufficient supply of autologous skin, such as when the third-degree burn area exceeds 30%, the skin donor site is already insufficient.

The skin is the largest organ of the human body, accounting for about 16% of the body weight, and is a natural barrier and a bridge of communication between the body and the outside world. The composition and structure of the skin are highly complex, including a variety of cells (epidermal cells, dermal cells, etc.), collagen fibers, elastic fibers, glycoproteins or proteoglycans and other biomacromolecules, as well as amino acids, glucose, vitamins and inorganic salts, etc. Small molecule. Human skin can be divided into four parts from the surface to the inside: the epidermis, basement membrane, dermis, and subcutaneous tissue. Among them, the epidermis and dermis are the most important components to realize skin function. The epidermis is an epithelial tissue with a thickness of about 0.1mm and is composed of about 10 layers of epidermal cells. The dermis is a dense connective tissue with a thickness of about 2-5mm, mainly composed of collagen fibers, elastic fibers and proteoglycans secreted by fibroblasts. The dermis is the most important structure for skin function. The complex composition and structure of the skin determines the diversity of its functions. The skin is first of all a natural barrier between the human body and the external environment. It can prevent the loss of water and electrolytes in the body, and can protect the body from the direct harm of radiation, chemicals, bacteria and viruses in the external environment. At the same time, the skin is the bridge between the body and the external environment. Through the metabolism of the skin, the balance of water and other substances inside the body can be adjusted, the body temperature can be adjusted, and the external information can be sensed.

Therefore, in the design of skin regeneration materials, the characteristics of the double-layer structure of the epidermis and dermis of the skin should usually be simulated, and the design has a double-layer structure that simulates the epidermis and dermis. Among them, the dermis scaffold plays the role of inducing skin dermis regeneration; while the epidermis covering plays the role of isolating the external environment, preventing bacterial invasion, and preventing water evaporation.

In my country, there are tens of millions of patients who need medical treatment due to burns, mechanical injuries or chronic skin ulcers every year. Therefore, the development of a new skin regeneration scaffold that can effectively promote wound healing and skin tissue regeneration has important social and economic benefits.

Contents of the invention

The purpose of the present invention is to provide a collagen-chitosan and silicone rubber double-layer skin regeneration support that can effectively promote wound healing and skin tissue regeneration and a preparation method thereof.

The collagen-chitosan and silicon rubber double-layer skin regeneration support of the present invention is composed of two parts: a collagen-chitosan porous support and a silicon rubber film.

The preparation method of collagen-chitosan and silicone rubber double-layer skin regeneration scaffold adopts freezing-lyophilization method, and the steps are as follows, and the following concentrations are all by mass:

1) Use acetic acid solution with a concentration of 0.3% to 5% to prepare collagen solution and chitosan solution with a concentration of 0.1% to 1%, respectively, drop the chitosan solution into the collagen solution, stir evenly, and vacuum defoam to obtain collagen - Chitosan mixed solution, wherein the content of chitosan solution is 5% to 50%;

2) injecting the collagen-chitosan blend into the mold, and freezing it into a solid at -5°C to -50°C by a freeze-drying method to obtain a collagen-chitosan three-dimensional porous scaffold;

3) The collagen-chitosan three-dimensional porous scaffold obtained in step 2) is first subjected to vacuum dry heat physical cross-linking treatment at a temperature of 80° C. to 130° C., and then cross-linked in a glutaraldehyde solution with a concentration of 0.05% to 1%. ~48 hours, after repeated washing, freeze and freeze-dry again;

4) Evenly coat the adhesive with good biocompatibility in the amount of 0.05-0.5mg/ ^cm2 on the surface of the silicone rubber film with a thickness of 0.05-0.5 mm, and then place the collagen-chitosan dermal scaffold on the coated adhesive The collagen-chitosan/silicone rubber bilayer skin regeneration scaffold was obtained on the surface of the silicone rubber film.

The above-mentioned adhesive with good biocompatibility may be α-cyanoacrylate adhesive, fibrin glue or gelatin. Among them, the α-cyanoacrylate adhesive can be selected from α-butyl cyanoacrylate or α-octyl cyanoacrylate. Said collagen is type I collagen, which can be bovine tendon collagen, pig skin collagen, or rat tail collagen.

Collagen-chitosan dermis support among the present invention, its outstanding feature is to take biodegradable collagen protein as main raw material, add a small amount of chitosan to promote cross-linking, strengthen and resist degradation; By controlling freeze-drying conditions, obtain Porous scaffolds with specific microstructures (pore size, porosity, etc.). In terms of material selection, natural materials such as collagen and chitosan from animal bodies have greater advantages than synthetic materials. Type I collagen is the main component of dermal extracellular matrix (ECM), and its molecular structure is rich in RGD peptide, which is the specific ligand of integrin (Integrin) on the cell membrane surface and has the function of promoting cell adhesion and growth. Type I collagen is rich in sources, easy to purify, and mild in immunogenicity. Chitosan is the product of deacetylation of chitin, which has the functions of anti-infection, pain relief, promoting blood coagulation and promoting wound healing, and is rich in sources. Adding a small amount of chitosan to the collagen can make up for the lack of amino groups in the collagen, thereby facilitating the cross-linking of the collagen and improving its degradation resistance and mechanical strength. Tests show that the scaffold can effectively promote the growth of fibroblasts and epidermal cells; the immunogenicity is mild and has good tissue compatibility; animal experiments show that it can be vascularized, that is, it can survive. This porous scaffold can be used as a temporary scaffold for tissue growth, and promotes the growth of wound granulation tissue, fibroblasts, endothelial cells and epidermal cells within the pores of the scaffold. These ingrown tissues or cells will secrete the corresponding extracellular matrix and then differentiate into new skin tissue, promoting the regeneration of damaged skin. At the same time, the exogenous collagen/chitosan porous membrane will gradually degrade with the continuous development of the new tissue, and be absorbed by the body, and finally the dermal tissue that is completely similar to the human body is obtained.

In the present invention, the silicone rubber film is selected as the simulant of the epidermis, because the silicone rubber has good biocompatibility, high air permeability and controllable moisture permeability, and has good elasticity and clinical ease of operation. These characteristics give the silicone rubber film the isolation function, the function of preventing bacterial invasion, and the function of preventing water evaporation, etc., so it can play the function of the epidermis, thereby ensuring the practicability of the double-layer skin scaffold.

The collagen-chitosan/silicone rubber composite porous scaffold of the present invention has the characteristics of a double-layer structure, wherein the collagen-chitosan porous scaffold layer can induce directional migration, proliferation and differentiation of wound cells, and induce regeneration of dermal tissue in situ. The silicone rubber film layer has functions such as preventing the volatilization of tissue moisture, keeping the wound moist, and preventing bacterial invasion. The biocompatible adhesive can realize the effective bonding of the collagen/chitosan porous scaffold layer and the silicone rubber film layer, which facilitates the operation of the operation and increases the practicability.

The invention provides a skin substitute with good performance for the treatment of patients with deep burns and chronic skin ulcers, which can significantly promote the healing of wounds, reduce the hyperplasia of scars, and further reduce the pain of patients. It can be widely used in burn wounds, plastic surgery and purpura eruptions. The preparation method of the invention is simple, the source of materials is wide, the production efficiency is high, and it is suitable for industrialized production. Compared with similar foreign products, the product of the present invention has great advantages in terms of cost.

Description of drawings

Figure 1 is a photo of the overall appearance of the collagen-chitosan/silicone rubber double-layer skin regeneration scaffold;

Fig. 2 is the microstructural diagram of collagen-chitosan/silicone rubber double-layer skin regeneration scaffold;

Fig. 3 is that gelatin is the collagen-chitosan/silicone rubber double-layer skin regeneration scaffold cytotoxic evaluation result of adhesive agent;

Fig. 4 is the cytotoxicity evaluation result of the collagen-chitosan/silicone rubber double-layer skin regeneration scaffold that octyl cyanoacrylate is an adhesive;

Fig. 5 is the general observation photo of collagen-chitosan/silicone rubber double-layer skin regeneration scaffold repairing the full-thickness skin defect wound on the pig's back after 4 weeks;

Fig. 6 is a tissue section diagram of the full-thickness skin defect wound on the pig's back 4 weeks after the collagen-chitosan/silicone rubber double-layer skin regeneration scaffold was repaired, and the scale bar is 200 microns.

Specific implementation method

Example 1:

Collagen and chitosan were dissolved in 3% acetic acid solution respectively, and the preparation concentration was 0.5% collagen solution and 0.5% chitosan solution, and the chitosan solution was dropped into the collagen solution, wherein the chitosan The content is 10%, stir evenly and vacuum degassing; use freeze-lyophilization method, freeze at -20°C for 2 hours, and then freeze-dry for 24 hours. Freeze-dried collagen/chitosan scaffolds were cross-linked in a vacuum oven at 105°C for 12 hours, cross-linked with 0.25% glutaraldehyde solution at 4°C for 24 hours, rinsed repeatedly with triple distilled water, and then frozen again - freeze-drying to obtain cross-linked collagen-chitosan porous scaffolds. On the surface of the silicone rubber film with a thickness of about 0.14 mm, evenly coat the gelatin adhesive with good biocompatibility in the amount of 0.25 mg/ ^cm2 on the surface of the silicone rubber film, and then gently place the collagen-chitosan porous scaffold On the surface of the silicone rubber film coated with gelatin, a collagen-chitosan/silicone rubber double-layer skin regeneration scaffold was obtained. Figure 1 and Figure 2 are the macroscopic topography and microstructure diagrams of the composite skin regeneration scaffold, respectively.

Example 2:

Collagen and chitosan were dissolved in 3% acetic acid solution respectively, and the preparation concentration was 0.5% collagen solution and 0.5% chitosan solution, and the chitosan solution was dropped into the collagen solution, wherein the chitosan The content is 10%, stir evenly and vacuum degassing; use freeze-lyophilization method, freeze at -20°C for 2 hours, and then freeze-dry for 24 hours. Freeze-dried collagen-chitosan scaffolds were cross-linked in a vacuum oven at 105°C for 12 hours, then cross-linked with 0.25% glutaraldehyde solution at 4°C for 24 hours, rinsed repeatedly with three-distilled water, and then frozen again - freeze-drying to obtain cross-linked collagen-chitosan porous scaffolds. On the surface of the silicone rubber film with a thickness of about 0.14 mm, evenly coat the α-cyanoacrylate octyl adhesive with good biocompatibility in the amount of 0.25 mg/ ^cm2 on the surface of the silicone rubber film, and then apply the collagen-shell The polysaccharide porous scaffold is gently placed on the surface of the gel-coated silicone rubber film to obtain a collagen-chitosan/silicone rubber double-layer skin regeneration scaffold.

Example 3:

Collagen and chitosan were dissolved in 0.3% acetic acid solution respectively to prepare a 0.5% collagen solution and a 0.5% chitosan solution, and the chitosan solution was dropped into the collagen solution, wherein the chitosan The content is 5%, stir evenly and vacuum degassing; use freeze-lyophilization method, freeze to solid at -5°C, and then freeze-dry for 24 hours. Freeze-dried collagen/chitosan scaffolds were cross-linked in a vacuum oven at 80°C for 12 hours, cross-linked with 0.05% glutaraldehyde solution at 4°C for 1 hour, rinsed repeatedly with triple distilled water, and then frozen again - freeze-drying to obtain cross-linked collagen-chitosan porous scaffolds. On the surface of the silicone rubber film with a thickness of about 0.05mm, the α-cyanoacrylate octyl adhesive with good biocompatibility is evenly coated on the surface of the silicone rubber film at an amount of 0.05mg/ ^cm2 , and then the collagen-shell The polysaccharide porous scaffold is gently placed on the surface of the gel-coated silicone rubber film to obtain a collagen-chitosan/silicone rubber double-layer skin regeneration scaffold.

Example 4:

Collagen and chitosan were dissolved in 3% acetic acid solution respectively, and the preparation concentration was 0.5% collagen solution and 0.5% chitosan solution, and the chitosan solution was dropped into the collagen solution, wherein the chitosan The content is 50%, stir evenly and vacuum degassing; use freeze-freeze drying method, freeze at -50°C for 2 hours, and then freeze-dry for 24 hours. Freeze-dried collagen-chitosan scaffolds were cross-linked in a vacuum oven at 130°C for 12 hours, then cross-linked with 1% glutaraldehyde solution at 4°C for 48 hours, rinsed repeatedly with triple distilled water, and then frozen again - freeze-drying to obtain cross-linked collagen-chitosan porous scaffolds. On the surface of the silicone rubber film with a thickness of about 0.14 mm, evenly coat the fibrin adhesive with good biocompatibility on the surface of the silicone rubber film at an amount of 0.5 mg/cm ² , and then gently apply the collagen-chitosan porous scaffold Placed on the surface of the silicone rubber film coated with glue, the collagen-chitosan/silicone rubber double-layer skin regeneration scaffold is obtained.

Example 5: Cytotoxicity of collagen-chitosan/silicone rubber double-layer skin regeneration scaffold with gelatin as adhesive

After the collagen-chitosan/silicone rubber double-layer skin regeneration scaffold obtained in Example 1 was sterilized by ethylene oxide, 10% calf serum, 100 units/ml penicillin and 100 units/ml streptavidin were added. The elemental DMEM culture solution was used as the leaching medium, and the leaching was carried out at 4°C for 24 hours. Using the extract as a culture medium, the activity of fibroblasts was observed over time ( FIG. 3 ), which proved that the double-layer skin regeneration scaffold had no cytotoxicity. Cell viability was detected by 3-(4,5-dimethylthiazole)-2,5-diphenyltetrazolium bromide (MTT) method.

Example 6: Cytotoxicity of collagen-chitosan/silicone rubber double-layer skin regeneration scaffold with α-cyanoacrylate octyl as adhesive

After the collagen-chitosan/silicone rubber double-layer skin regeneration scaffold obtained in Example 2 was sterilized by ethylene oxide, 10% calf serum, 100 units/ml penicillin and 100 units/ml streptavidin were added. The elemental DMEM culture solution was used as the leaching medium, and the leaching was carried out at 4°C for 24 hours. Using the extract as a culture medium, the activity of fibroblasts was observed over time ( FIG. 4 ), which proved that the double-layer skin regeneration scaffold had no cytotoxicity. Cell viability was detected by 3-(4,5-dimethylthiazole)-2,5-diphenyltetrazolium bromide (MTT) method.

Example 7: In vitro skin regeneration ability evaluation of collagen-chitosan/silicone rubber double-layer skin regeneration scaffold

Prepare the cross-linked collagen-chitosan scaffold with Example 1. On a silicone rubber film with a thickness of about 0.14 mm, evenly coat the α-cyanoacrylate octyl adhesive with good biocompatibility on the surface of the silicone rubber film at an amount of 0.5 mg/cm ² , and then coat the collagen/chitopolymer The sugar porous scaffold is gently placed on the surface of the gel-coated silicone rubber film to obtain a collagen-chitosan/silicone rubber double-layer skin regeneration scaffold. The composite regenerative scaffold was sterilized by ethylene oxide, transplanted to the full-thickness skin defect on the back of the pig, and routinely packed and bandaged. The wound healing was evaluated 3, 7, 14, and 28 days after the operation. Figure 5 is the general observation of the wound 4 weeks after the transplantation, and Figure 6 is a tissue slice picture 4 weeks after the transplantation, which proves that the dermal tissue has grown into the scaffold.

Claims (4)

1. collagen-chitin and silicon rubber bilayer skin regeneration support is characterized in that being made up of collagen-chitin porous support and silicon rubber film two parts, prepare as follows, following concentration all by mass:

1) with concentration be 0.3%～5% acetic acid solution respectively compound concentration be 0.1%～1% collagen solution and chitosan solution, chitosan solution is splashed in the collagen solution, the vacuum defoamation that stirs, the collagen-chitin mixed solution, wherein the content of chitosan solution is 5%～50%;

2) the collagen-chitin blended liquid is injected mould, adopt the freeze-dried method, be frozen into solid under-5 ℃～-50 ℃ temperature, lyophilizing obtains the collagen-chitin three-dimensional porous rack;

3) with step 2) the collagen-chitin three-dimensional porous rack of gained earlier under 80 ℃～130 ℃ temperature the xeothermic physical crosslinking of vacuum handle, be in 0.05%～1% glutaraldehyde solution crosslinked 1～48 hour in concentration again, after cleaning repeatedly, freezing once more, lyophilizing;

4) biocompatibility is good adhesive is with 0.05～0.5mg/cm ²Amount evenly to be coated in thickness be 0.05～0.5 millimeter silicon rubber film surface, then the collagen-chitin dermis scaffold is placed on the silicon rubber film surface of adhesive coating, obtain collagen-chitin/silicon rubber bilayer skin regeneration support.

2. the preparation method of collagen-chitin according to claim 1 and silicon rubber bilayer skin regeneration support is characterized in that comprising the steps, following concentration all by mass:

1) with concentration be 0.3%～5% acetic acid solution respectively compound concentration be 0.1%～1% collagen solution and chitosan solution, chitosan solution is splashed in the collagen solution, the vacuum defoamation that stirs, the collagen-chitin mixed solution, wherein the content of chitosan solution is 5%～50%;

2) the collagen-chitin blended liquid is injected mould, adopt the freeze-dried method, be frozen into solid under-5 ℃～-50 ℃ temperature, lyophilizing obtains the collagen-chitin three-dimensional porous rack;

3) with step 2) the collagen-chitin three-dimensional porous rack of gained earlier under 80 ℃～130 ℃ temperature the xeothermic physical crosslinking of vacuum handle, be in 0.05%～1% glutaraldehyde solution crosslinked 1～48 hour in concentration again, after cleaning repeatedly, freezing once more, lyophilizing;

4) biocompatibility is good adhesive is with 0.05～0.5mg/cm ²Amount evenly to be coated in thickness be 0.05～0.5 millimeter silicon rubber film surface, then the collagen-chitin dermis scaffold is placed on the silicon rubber film surface of adhesive coating, obtain collagen-chitin/silicon rubber bilayer skin regeneration support.

3. the preparation method of collagen-chitin according to claim 2 and silicon rubber bilayer skin regeneration support is characterized in that the good adhesive of said biocompatibility is a-cyanoacrylate class adhesive, Fibrin Glue or gelatin.

4. the preparation method of collagen-chitin according to claim 3 and silicon rubber bilayer skin regeneration support is characterized in that a-cyanoacrylate class adhesive selects alpha-cyanoacrylate butyl ester or alpha-cyanoacrylate monooctyl ester for use.

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