Artificial dermis repair material and preparation method thereof

Abstract

The invention discloses an artificial dermal repair material and a preparation method thereof, the artificial dermal repair material adopts fresh pigskin as a raw material, chitosan is introduced for compounding, physical, chemical and biochemical methods are ingeniously used, and an acellular dermal matrix with good biocompatibility, adhesion, moisture retention and air permeability is prepared. The product has better physical properties and antibacterial and bacteriostatic effects, and overcomes the defect that the existing clinical artificial dermis has no antibacterial and bacteriostatic effects.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and particularly relates to an artificial dermis repair material and a preparation method thereof. Background Art

[0002] The skin is the largest organ in the human body with advanced organizational structures, and has functions such as protection, sensation, body temperature regulation, secretion and excretion, absorption, material metabolism, and immunity. Autologous skin transplantation, as the gold standard for the treatment of various traumatic skin defects including burns and senile diseases, has achieved great success in clinical practice. However, for patients with large-area skin defects, especially severe burn patients, the shortage of autologous skin sources has always restricted the treatment process of the patients.

[0003] An artificial dermis scaffold material needs to form in the human body with good biocompatibility, degradability, and antibacterial properties, which is conducive to the adhesion of skin stem cells, and the growth and reproduction on its surface, providing a good microenvironment for skin stem cells. An appropriate scaffold material plays an extremely important role in the repair process of artificial dermis wounds.

[0004] Although artificial dermis is increasingly widely used, however, susceptibility to infection is an inevitable defect. Since the artificial dermis fails to establish a reliable blood supply with the wound surface in the early stage after transplantation, its antibacterial ability is poor, and the infection rate after transplantation is relatively high. Once an infection occurs, it will not only lead to the failure of transplantation, but even threaten the life of the patient. Summary of the Invention

[0005] The purpose of the present invention is to provide an artificial dermis repair material and a preparation method thereof. It uses fresh pig skin as a raw material, and skillfully uses physical, chemical, and biochemical methods to prepare a decellularized dermal matrix with good biocompatibility, adhesiveness, moisture retention, and air permeability. This product has good physical properties and antibacterial and bacteriostatic effects, solving the defect that current clinical artificial dermis has no antibacterial and bacteriostatic effects.

[0006] To achieve the above purpose, the technical solutions adopted by the present invention are as follows.

[0007] The artificial dermis repair material involved in the present invention is obtained by compounding a decellularized dermal matrix and chitosan.

[0008] The preparation method of the decellularized matrix dermal matrix includes the following steps.

[0009] (1) Pretreatment: Select fresh pig skin, depilate and disinfect the skin-taking area, and cut it into thin slices.

[0010] (2) Degreasing: Use an organic solvent for extraction to remove fat and fat-soluble impurities.

[0011] (3) Decellularization: Use a 0.1 - 0.5% surfactant solution in combination with a 0.1 - 0.5% protease solution, and perform ultrasonic treatment for 6 - 12 h.

[0012] (4) Antigen removal: Under the condition of pH 7 - 8, react the surfactant with the acellular porcine skin material for 30 - 40 hours, where the active reagent is at least one of small molecule organic acid anhydrides, acyl chlorides, amides, epoxides, and haloalkanes; then add a strong hydrogen bond reagent (such as guanidine compounds) and react for 30 - 40 hours to obtain the membrane material tissue after complete antigen removal.

[0013] (5) Crosslinking: Add the above - obtained porcine skin material into a non - aldehyde crosslinking agent solution. The non - aldehyde crosslinking agent is at least one of epoxides, diisocyanates, polyethylene glycols, and carbodiimides; the mass concentration of the non - aldehyde crosslinking agent is 1.0 - 4.0%. React under the conditions of pH 7.0 - 8.0 and stirring for 2 - 8 days.

[0014] (6) Chitosan compounding: Prepare a chitosan solution with a concentration of 5 - 15 g / L, stir and dissolve it into a uniform solution in a water bath at 40 - 60 °C, and set it aside after cooling. Immerse the crosslinked acellular dermal matrix obtained in step (5) in the chitosan solution, and stir for 1 - 2 h to mix evenly.

[0015] (7) Compound freeze - drying: Place the processed acellular dermal matrix on a mold and perform vacuum freeze - drying for 24 - 48 h to obtain the artificial dermal repair material.

[0016] The beneficial effect of the present invention is that chitosan is the product of chitin deacetylation. Animal studies have confirmed that it can act on epidermal cells and promote wound epithelialization; it can combine with heparin, PDGF, etc. in plasma to form polyelectrolyte complexes and stimulate the growth of fibroblasts. Adding chitosan can reduce the degradation rate of collagen and increase its strength. At the same time, chitosan also has a broad - spectrum antibacterial and antifungal effect, which is beneficial to solving the problem of easy infection when the artificial dermal material is applied to wound repair. Detailed implementation mode

[0017] The following will clearly and completely describe the concept and the resulting technical effects of the present invention in combination with the embodiments to fully understand the purpose, features, and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts all belong to the scope of protection of the present invention.

[0018] Example 1

[0019] This embodiment provides an artificial dermis repair material, and its preparation process is as follows: (1) Pretreatment: Clean the fresh pigskin, disinfect it, remove villi, fibers, large adipose tissues, etc., and cut it into thin slices with a slicing machine. (2) Degreasing: Use organic solvents for extraction to remove fats and fat-soluble impurities. (3) Decellularization: Use a 0.1% surfactant solution in combination with a 0.5% protease solution to ultrasonically treat the pigskin material. (4) Deantigenization: Use a reactive reagent to react with the pigskin for 12 hours at a pH value of 7, and then use a strong hydrogen bond reagent to react with the pigskin for 12 hours at a pH value of 7 to obtain a deantigenized and decellularized dermal matrix material. (5) Crosslinking: Use 1% carbodiimide to react with the above material for 1 day under stirring at pH 9. (6) Chitosan compounding: Prepare a chitosan solution with a concentration of 10 g / L, stir and dissolve it into a uniform solution in a water bath at 50 °C, cool it and set it aside. Immerse the crosslinked decellularized dermal matrix obtained in step (5) in the chitosan solution, stir for 1 - 2 h, and mix evenly. (7) Compound freeze-drying: Vacuum freeze-dry the crosslinked decellularized dermal matrix to obtain the artificial dermis repair material.

[0020] Example 2

[0021] This embodiment is a modification of Example 1. The step of chitosan compounding after crosslinking is removed, and the crosslinked decellularized dermal matrix is directly freeze-dried to obtain the artificial dermis repair material.

[0022] Example 3

[0023] This embodiment provides a verification of the performance effect of an artificial dermis repair material: Cut the artificial dermis repair material samples containing chitosan solutions with different concentrations into 6-mm round pieces, use the artificial dermis repair material without chitosan solution as a control, and sterilize them by ultraviolet irradiation for 30 min in a laminar flow hood for later use.

[0024] Prepare MH agar medium according to the instructions, heat it to completely dissolve, sterilize it in a steam sterilizer at 121 °C for 15 min. When the temperature drops to 40 - 55 °C, pour it into petri dishes in a sterile environment and let it cool and solidify for later use.

[0025] Dip a sterile cotton swab into the Pseudomonas aeruginosa and Staphylococcus aureus bacterial suspensions with a calibrated 0.5 McFarland turbidity, and gently lean against the tube wall to squeeze out the excess bacterial suspension. Use the cotton swab to coat the entire surface of the culture medium, rotate the petri dish 60 degrees each time, and smear three times. Finally, wipe around the perimeter twice to ensure even spreading of the bacterial suspension. Use sterile forceps to attach the sample to the surface of the petri dish, and do not pick it up again after attachment. Flip the petri dish and incubate it in a 37°C constant temperature incubator for 18 - 24 hours, then take it out for measurement. Use a ruler to measure the diameter of the inhibition zone on the back of the petri dish (measure once horizontally and vertically and take the average, and round the average to an integer). Set up three parallel groups for each petri dish. The edge of the inhibition zone is defined as the limit where no obvious bacterial growth can be seen with the naked eye. The experimental results show that the prepared artificial dermis repair material basically shows an inhibition zone, indicating its antibacterial function. The inhibition zone results suggest that compared with Staphylococcus aureus, the artificial dermis repair material has a more obvious antibacterial effect on Pseudomonas aeruginosa.

Claims

1. A method for preparing an artificial dermis repair material, characterized in that: The following steps are involved: (1) Pretreatment: Select fresh pig skin, depilate and disinfect the skinning area, and cut into thin slices; (2) Degreasing: Use organic solvents to extract and remove fat and fat-soluble impurities; (3) Decellularization: Use 0.1-0.5% surfactant solution combined with 0.1-0.5% protease solution and ultrasonic treatment for 6-12h; (4) Antigen removal: At a pH of 7-8, an active agent is reacted with the decellularized pig skin material for 30-40 hours, wherein the active agent is at least one of a small molecule organic acid anhydride, an acyl chloride, an amide, an epoxide and a methyl halide; then a strong hydrogen bonding agent (such as a guanidine compound) is added and reacted for 30-40 hours to obtain a membrane tissue after the antigen is completely removed; (5) Cross-linking: Add the above-obtained pigskin material to a non-aldehyde cross-linking agent solution, wherein the non-aldehyde cross-linking agent is at least one of epoxide, diisocyanate, polyethylene glycol and carbodiimide; the mass concentration of the non-aldehyde cross-linking agent is 1.0-4.0%. The reaction is carried out for 2-8 days under stirring at a pH value of 7.0-8.0; (6) Chitosan compounding: prepare chitosan solution with a concentration of 5-15 g / L, stir and dissolve in a 40-60°C water bath to form a uniform solution, cool and set aside, soak the cross-linked acellular dermal matrix obtained in step (5) in the chitosan solution, stir for 1-2 hours, and mix evenly; (7) Composite freeze-drying: Place the processed decellularized dermal matrix on a mold and vacuum freeze-dry for 24-48 hours to obtain artificial dermal repair material.

2. The method for preparing the artificial dermis repair material according to claim 1, characterized in that: The raw material selected is fresh pig skin.

3. The method for preparing the artificial dermis repair material according to claim 1, characterized in that: The non-aldehyde chemical cross-linking agent is at least one of epoxide, diamide, diisocyanate, polyethylene glycol and carbodiimide.

4. The method for preparing the artificial dermis repair material according to claim 3, characterized in that: The concentration of the non-aldehyde chemical cross-linking agent is 1.0-4.0%.

5. The method for preparing the artificial dermis repair material according to claim 1, characterized in that: The deacetylation degree of the chitosan solution is ≥85%.

6. The method for preparing the artificial dermis repair material according to claim 5, characterized in that: The concentration of the chitosan solution is 5-15 g / L.

7. The method for preparing an artificial dermis repair material according to claim 1, characterized in that: When the chitosan concentration is 5-15g / L, the bactericidal rates of chitosan composite artificial dermis repair materials against Staphylococcus aureus and Pseudomonas aeruginosa are close to or greater than 80%.

8. The artificial dermis repair material obtained by the preparation method of the artificial dermis repair material according to claim 1, characterized in that: The artificial dermis repair material is prepared by the method for preparing an artificial dermis repair material according to any one of claims 1 to 7.