Porous moist artificial skin as well as preparation method and application thereof

Abstract

The invention relates to porous moist artificial skin as well as a preparation method and application thereof. Specifically, a freeze-dried film with a continuous microporous structure is prepared by a freeze-drying method, and then the freeze-dried film is subjected to infiltration treatment by a gel solution, so that the freeze-dried film has relatively high flexibility. The porous moist artificial skin has the characteristics of being porous and moist, wound cells can be attached to the porous moist artificial skin, a moist environment is created at a wound, wound healing is promoted, meanwhile, the porous moist artificial skin has high flexibility and can better adapt to different wound shapes such as joints and large wound undulation degree, and the application range of the porous moist artificial skin is widened. Therefore, the dressing can be better combined with the wound to promote wound healing. In addition, the skin repairing gel also has the multifunctional characteristics of resisting bacteria, diminishing inflammation, reducing scar area and the like, has high wound combination degree, and can provide higher practicability for the fields of clinical skin repairing and skin transplantation.

Description

Technical Field

[0001] The invention belongs to the field of biomedical engineering and material design and regulation, and relates to a porous moist artificial skin and a preparation method and application thereof. Background Art

[0002] As the largest organ in the human body, the skin acts as a barrier, buffering and protecting internal organs and isolating harmful substances and bacteria from the body. Burns, diabetic foot, bedsores, contusions, etc. can cause large-area skin wounds. Such wounds have a long healing period, and the wound exudates are prone to bacterial infection and induce inflammation, which seriously affect the healing of wounds. In addition, the commonly used dressings for wounds at joints or wounds with large morphological fluctuations, such as artificial skin prepared by freeze-drying, are brittle and poorly flexible, and are not highly integrated with complex skin wounds and cannot effectively promote wound healing.

[0003] Therefore, there is an urgent need for a wound dressing that has good flexibility, good adhesion to complex wounds, and can effectively promote wound healing. Summary of the invention

[0004] The prior art lacks wound dressings that are flexible, well integrated with complex wounds, and can effectively promote wound healing. Based on the above problems, in order to better adapt to different wound morphologies, the purpose of this application is to provide a porous moist artificial skin and its preparation method and application.

[0005] The present invention regulates the structure and physical and chemical properties of artificial skin by infiltrating the freeze-dried gel film, and the key is to prepare porous moist artificial skin by infiltrating the freeze-dried film with gel solution of different concentrations. The porous moist artificial skin is an artificial skin dressing with high toughness, which can adapt to different wound morphologies, better combine with the wound, and has excellent effects of promoting wound healing and protecting the wound.

[0006] The freeze drying method involved in the present application is a simple and effective method, which can be used to prepare a gel film with a large specific surface area and a porous structure. The pore size of the porous gel prepared by the freeze drying method is mostly 20 to 150 μm. When applied to the preparation of artificial skin, a breathable environment can be created, and it has a certain strength, which can protect the wound and promote the recovery of the wound. On the basis of the freeze-dried film, the gel liquid freeze-dried film is used as a matrix and is soaked in an appropriate amount of gel liquid again, which can improve the brittle and hard characteristics of the freeze-dried film, thereby obtaining an artificial skin with porous moisture, which helps to create a moist and sterile environment at the wound and has a better bonding degree for wounds with large surface undulations. Therefore, the artificial skin prepared by the present invention is an artificial skin with excellent prospects.

[0007] The present invention provides a wet and porous artificial skin obtained by treating a freeze-dried film with gel solution of different concentrations. The technical problem to be solved by the present invention is to provide a porous and moist artificial skin and a manufacturing method thereof. The artificial skin has better flexibility than the artificial skin prepared by freeze-drying method, can create a moist and sterile environment at the wound to promote wound healing and can adapt to wounds of different morphologies, and has antibacterial, hemostatic and anti-inflammatory multifunctional properties, and has good comprehensive performance.

[0008] The technical solution of the present invention includes:

[0009] One of the technical solutions of the present invention provides a porous moist artificial skin, which is a freeze-dried membrane soaked in a gel solution; the freeze-dried membrane has a pore size of 20 to 150 μm and is prepared as a matrix from a natural polymer compound, a cross-linking agent, an antibacterial factor and / or a hemostatic factor and / or an anti-inflammatory factor by cross-linking and freeze-drying; the gel solution is a solution prepared from the natural polymer compound, the cross-linking agent, the antibacterial factor and / or the hemostatic factor and / or the anti-inflammatory factor.

[0010] Furthermore, the natural polymer compound is any one or more of cellulose, starch, alginate, chitosan, hyaluronic acid, chondroitin sulfate, heparin, dextran, collagen, serum protein, soy protein, zein, casein, chitin and gelatin;

[0011] Furthermore, the cross-linking agent is one or more of catechin, gallic acid, tannic acid, isocyanate, zinc chloride, cellulose, polyurethane, chlorogenic acid, ferulic acid, protocatechuic aldehyde, syringic acid, vanillic acid, and p-hydroxybenzoic acid;

[0012] Further, the antibacterial factor is at least one of gallium nitrate, silver chloride, zinc nitrate, cupric chloride and ferric chloride;

[0013] Furthermore, the hemostatic factor is at least one of collagen, gelatin sponge, cellulose material, polyacrylamide, and chitosan;

[0014] Furthermore, the anti-inflammatory factor is at least any one of hyaluronic acid, chitosan, curcumin, β-glucan and propolis.

[0015] In some specific embodiments of the present application, the natural polymer compound is selected from carboxymethyl chitosan CMC and gelatin Gel, the cross-linking agent is selected from protocatechuic aldehyde PCA, and the antibacterial factor is selected from hydrated gallium nitrate.

[0016] Furthermore, the natural polymer compounds in the freeze-dried film provide it with excellent flexibility; the cross-linking agent in the freeze-dried film is used to construct a cross-linked network system to form a cross-linked network; the antibacterial factor and / or hemostatic factor and / or anti-inflammatory factor are used to provide antibacterial and / or hemostatic and / or anti-inflammatory properties.

[0017] The matrix of the present application adopts a porous freeze-dried film, which mainly serves as a structural skeleton to enhance the structural strength of the artificial skin, absorb wound exudate and promote cell proliferation and adhesion. The freeze-dried film is soaked in a natural polymer compound, which mainly serves to make the freeze-dried film have high flexibility and can adapt to different wound morphologies. This artificial skin dressing with high toughness can better adapt to different wound morphologies and closely combine with the wound to promote wound healing more efficiently, and can well meet the application in chronic wounds, contusions, diabetic foot and other aspects.

[0018] The second technical solution of the present invention also provides a method for preparing a porous moist artificial skin, comprising the following steps:

[0019] (1) dissolving a natural polymer compound in a solvent, heating and stirring in a water bath to dissolve the compound, and obtaining a compound solution; simultaneously dissolving a cross-linking agent, an antibacterial factor, and / or a hemostatic factor, and / or an anti-inflammatory factor in the solvent, adjusting the pH to 9 to 10, and stirring to form a complex solution;

[0020] (2) adding the complex solution to the compound solution to obtain a mixed solution, heating and stirring in a water bath until a gel is formed, and freeze-drying the gel product to form a freeze-dried film;

[0021] (3) Using the freeze-dried film obtained in step (2) as a matrix, immersing it in a gel solution, and finally obtaining an artificial skin with high flexibility after cooling, i.e., the porous moist artificial skin.

[0022] The solvent described in step (1) is any one of deionized water and PBS.

[0023] The mass concentration of the compound solution described in step (1) is 2-6%; the concentration of the cross-linking agent in the complex solution is 4-5%, the concentration of the antibacterial factor and / or hemostatic factor and / or anti-inflammatory factor is 2-3%, and the molar mass ratio of the cross-linking agent, antibacterial factor and / or hemostatic factor and / or anti-inflammatory factor is 3:1.

[0024] The pH adjustment in step (1) is achieved by adding a sodium hydroxide solution dropwise.

[0025] The temperature of the water bath heating in step (1) or (2) is 45-55°C, preferably 50°C.

[0026] The mass ratio of the complex solution in the mixed solution of step (2) to the solute in the compound solution is (2-5):20.

[0027] The gel formation in step (2) is a cross-linking process, and the cross-linking method is chemical cross-linking.

[0028] In step (3), the mass percentage of the natural polymer compound in the gel solution is 2%, the concentration of the cross-linking agent is 4-5%, and the concentration of the antibacterial factor and / or hemostatic factor and / or anti-inflammatory factor is 2-3%.

[0029] The freeze-dried film in step (3) is immersed in the gel solution for 5 to 10 minutes.

[0030] The cooling in step (3) is to place the freeze-dried film immersed in the gel solution in a refrigerator to lower the temperature.

[0031] A third technical solution of the present invention provides an application of the porous moist artificial skin described above, wherein the porous moist artificial skin is used as a wound dressing during wound treatment to create a sterile environment and promote wound healing.

[0032] The present invention has the following characteristics:

[0033] 1) The treated artificial skin has the characteristics of being porous and moist, which can create a moist and sterile environment at the wound to promote wound healing;

[0034] 2) The treated artificial skin has strong flexibility and can produce large deformation, so it can adapt to different wound morphologies, such as wounds at joints or wounds with large undulations, can have better wound adhesion, and has a certain strength to prevent the wound from being damaged again;

[0035] 3) The porous and moist gel membrane can serve as a temporary artificial skin and promote the self-repair of human skin;

[0036] 4) The invention adopts freeze-drying and soaking methods, which have the advantages of convenient and mature preparation process, simple operation, and large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The schematic diagram of the structure of the present invention; wherein ① is a freeze-dried film, ② is a gel solution, and ③ is an artificial skin with high toughness after infiltration;

[0038] Figure 2 The transmittance test of 2% CMC / Gel freeze-dried film was conducted by soaking it in 2% gel solution;

[0039] Figure 3 This is a schematic diagram of the lyophilized membranes of CMC / Gel with different concentrations immersed in a 2% gel solution;

[0040] Figure 4 The graph shows the antibacterial rate of artificial skin plate counts made by infiltrating freeze-dried CMC / Gel films of different concentrations in a 2% gel solution. DETAILED DESCRIPTION

[0041] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is described in detail below in conjunction with specific embodiments. It should be noted that the following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that for those of ordinary skill in the art, several variations and improvements can also be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0042] All raw materials of the present invention have no particular limitation on their sources, and can be purchased from the market or prepared according to conventional methods known to those skilled in the art.

[0043] Example 1 A porous moist artificial skin with carboxymethyl chitosan / gelatin freeze-dried film as the matrix material. This example uses PCA@Ga cross-linked carboxymethyl chitosan with a pore size of 20 to 150 μm and gelatin freeze-dried film as the matrix, carboxymethyl chitosan and gelatin solution as the gel solution, and the freeze-dried film is immersed in the gel solution to prepare high-toughness artificial skin. Two natural polymer compounds are stirred evenly in deionized water to obtain a mixed solution, and the prepared PCA@Ga complex solution is slowly added dropwise to the mixed solution, heated in a water bath at 50°C and stirred to form a gel, and then freeze-dried to form a porous freeze-dried film, and the freeze-dried film is immersed in 2% mixed solution to prepare an artificial skin with high toughness (refer to the schematic diagram of the process). Figure 1 ). The aldehyde group in the molecular structure of protocatechuic aldehyde (PA) can form Schiff base bonds with the amino groups on CMC and Gel. The PA@Ga complex acts as a crosslinking point to connect the CMC and Gel molecular chains together to form a dynamic crosslinking network. Among the crosslinking agent raw materials, PA and gallium nitrate both play a role in promoting crosslinking, among which protocatechuic aldehyde promotes crosslinking by forming Schiff base bonds, and gallium nitrate promotes crosslinking by forming metal coordination, and sodium hydroxide plays a role in adjusting the pH of the crosslinking agent.

[0044] The specific steps include:

[0045] (1) 0.2 g of carboxymethyl chitosan CMC and 0.2 g of gelatin Gel were dissolved in 10 mL of deionized water, respectively, and heated to dissolve in a 50° C. water bath, and then mixed in a mass ratio of carboxymethyl chitosan CMC to gelatin Gel of 1:1 to obtain a compound solution with a concentration of 2%;

[0046] (2) 0.41436 g (3 mmol) of protocatechuic aldehyde PCA and 0.25574 g (1 mmol) of hydrated gallium nitrate were dissolved in 10 mL of deionized water at a molar mass ratio of 3:1, and the pH was adjusted to 9-10 with sodium hydroxide to form a PCA@Ga complex to obtain a complex solution;

[0047] (3) slowly dropping the PCA@Ga complex solution into the compound solution prepared in step (1), wherein the solute mass ratio of the PCA@Ga complex solution to the compound solution is 3g:20g, and after obtaining the mixed solution, heating in a water bath at 50°C and stirring to form a gel, placing the gel in a refrigerator for 1 day, and then freeze-drying to form a freeze-dried film;

[0048] (4) The freeze-dried film is immersed in a gel solution for 10 minutes, wherein the mass concentration of CMC / Gel in the gel solution is 2%, and the mass concentration of the PCA@Ga complex is 0.3%, that is, the mass ratio of the PCA@Ga complex to CMC / Gel is 3:20, thereby obtaining an artificial skin with high toughness, namely, a porous moist artificial skin.

[0049] Example 2 A porous moist artificial skin with carboxymethyl chitosan / gelatin freeze-dried film as the base material

[0050] The steps of this embodiment are basically the same as those of embodiment 1, except that the concentration of the compound solution in step (1) is replaced with a mass concentration of 3%.

[0051] Example 3 A porous moist artificial skin with carboxymethyl chitosan / gelatin freeze-dried film as the base material

[0052] The steps of this embodiment are basically the same as those of embodiment 1, except that the concentration of the compound solution in step (1) is replaced with a mass concentration of 4%.

[0053] The porous moist artificial skin prepared in the above Examples 1 to 3 was characterized in terms of performance, including transmittance, flexibility and antibacterial rate, wherein the antibacterial rate involved a plate antibacterial test, and the specific operating steps are as follows:

[0054] 1. Experimental materials:

[0055] Preparation of culture medium: The culture medium used is LB culture medium + nutrient agar culture medium; Sample preparation: Cut and sterilize in advance; Preparation of consumables: sterile pipette tips (5mL, 1mL, 200μL), sterile centrifuge tubes (2mL, 15mL, 50mL), sterile conical flasks, sterile disposable culture dishes; LB culture medium and agar plates can be prepared in advance and placed in the clean bench for a few days before use; Prepare a sufficient amount of sterilized PBS in advance; The bacteria involved in this experiment are Escherichia coli and Bacillus subtilis.

[0056] 2. Experimental process:

[0057] Day 1

[0058] The bacteria were inoculated into a conical flask containing 25 mL of LB medium using an inoculation loop and placed in a shaker overnight (37° C., 60 rpm, 18-24 h).

[0059] Day 2

[0060] (1) Place the material into a well plate, wash it twice with PBS, and remove excess liquid.

[0061] (2) Cultivate the bacterial solution in a shaking incubator overnight until the density is about 10 8 / mL, need to be diluted to 10 5 / mL:

[0062] Dilute to 10% with LB medium 7 / mL (1mL bacterial solution + 9mL LB medium), vortex to disperse;

[0063] Dilute to 10 with PBS 5 / mL, vortex to disperse.

[0064] (3) Add 0.2 mL of 10 5 / mL of bacterial solution, seal the well plate with sealing film and shake on a shaker for 12-18h (25℃, 120rpm).

[0065] Day 3

[0066] (1) Elution: Transfer the material to a 10 mL centrifuge tube and add 10 mL PBS for elution (the default density is 10 4 / mL);

[0067] Dilute to 10 with PBS 3 / mL and 10 2 / mL (use 1.5mL ep tube, 0.1mL bacterial solution + 0.9mL PBS).

[0068] (2) Take 100 μL of density 10 3 / mL and 10 2 / mL of bacterial solution were spread on plates and cultured in an incubator (37°C).

[0069] Day 4

[0070] Observe the colonies produced on the agar plate, take photos and count them.

[0071] Characterization and experimental results reference Figure 2 to Figure 4 .

[0072] Figure 1It means that the gel liquid of different concentrations after chemical cross-linking is freeze-dried to prepare a matrix with a porous structure, and then the freeze-dried film with a porous structure after freezer drying is immersed in the gel liquid again to prepare an artificial skin with porous and moist characteristics.

[0073] Figure 2 This is a light transmittance test diagram of an embodiment of the present invention using a 2% CMC / Gel freeze-dried film as a substrate immersed in a 2% gel solution. Figure 2 It can be seen that the 2% CMC / Gel freeze-dried film immersed in 2% gel solution has a porous structure and high light transmittance.

[0074] Figure 3 This is a real picture of a 4% CMC / Gel freeze-dried film immersed in a 2% gel solution. Figure 3 It can be seen that the artificial skin prepared by soaking the 4% CMC / Gel freeze-dried film in 2% gel solution has good flexibility and can still return to its original shape after bending. It can be applied to joints or wounds with large undulations.

[0075] Figure 4 The plate count inhibition rate diagram of the freeze-dried membranes of different concentrations in the present invention immersed in 2% gel solution is shown in Figure 2. Figure 4 It can be seen that the artificial skin prepared by infiltrating different concentrations of freeze-dried films in 2% concentration gel solution has good antibacterial properties against Escherichia coli (first row, Gram-negative bacteria) and Bacillus subtilis (second row, Gram-positive bacteria).

[0076] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A porous moist artificial skin, characterized in that: It is a freeze-dried membrane infiltrated with gel solution; The freeze-dried membrane has a pore size of 20 to 150 μm and is prepared as a matrix from natural polymer compounds, cross-linking agents, antibacterial factors and / or hemostatic factors and / or anti-inflammatory factors through cross-linking and freeze-drying; the gel solution is a solution prepared from natural polymer compounds, cross-linking agents, antibacterial factors and / or hemostatic factors and / or anti-inflammatory factors.

2. A porous moist artificial skin according to claim 1, characterized in that: The natural high molecular compound is any one or more of cellulose, starch, alginate, chitosan, hyaluronic acid, chondroitin sulfate, heparin, dextran, collagen, serum protein, soybean protein, zein, casein, chitin and gelatin.

3. A porous moist artificial skin according to claim 1, characterized in that: The cross-linking agent is one or more of catechin, gallic acid, tannic acid, isocyanate, zinc chloride, cellulose, polyurethane, chlorogenic acid, ferulic acid, protocatechuic aldehyde, syringic acid, vanillic acid, and p-hydroxybenzoic acid.

4. A porous moist artificial skin according to claim 1, characterized in that: The antibacterial factor is at least one of gallium nitrate, silver chloride, zinc nitrate, copper chloride, and ferric chloride; the hemostatic factor is at least one of collagen, gelatin sponge, cellulose material, polyacrylamide, and chitosan; the anti-inflammatory factor is at least one of hyaluronic acid, chitosan, curcumin, β-glucan, and propolis.

5. The method for preparing a porous moist artificial skin as claimed in claim 1, characterized in that: The steps include: (1) dissolving a natural polymer compound in a solvent, heating and stirring in a water bath to dissolve the compound, and obtaining a compound solution; simultaneously dissolving a cross-linking agent, an antibacterial factor, and / or a hemostatic factor, and / or an anti-inflammatory factor in the solvent, adjusting the pH to 9 to 10, and stirring to form a complex solution; (2) adding the complex solution to the compound solution to obtain a mixed solution, heating and stirring in a water bath until a gel is formed, and freeze-drying the gel product to form a freeze-dried film; (3) Using the freeze-dried film obtained in step (2) as a matrix, immersing it in a gel solution, and finally preparing a porous moist artificial skin after cooling.

6. The method for preparing a porous moist artificial skin according to claim 5, characterized in that: The solvent described in step (1) is any one of deionized water and PBS; the mass concentration of the compound solution is 2-6%; the concentration of the cross-linking agent in the complex solution is 4-5%, the concentration of the antibacterial factor and / or hemostatic factor and / or anti-inflammatory factor is 2-3%, and the molar mass ratio of the cross-linking agent, antibacterial factor and / or hemostatic factor and / or anti-inflammatory factor is 3:

1.

7. The method for preparing a porous moist artificial skin according to claim 5, characterized in that: The mass ratio of the complex solution in the mixed solution of step (2) to the solute in the compound solution is (2-5):20; the temperature of the water bath heating in step (1) or (2) is 45-55°C.

8. The method for preparing a porous moist artificial skin according to claim 5, characterized in that: The mass percentage of the natural polymer compound in the gel solution of step (3) is 2%, the concentration of the cross-linking agent is 4-5%, and the concentration of the antibacterial factor and / or hemostatic factor and / or anti-inflammatory factor is 2-3%.

9. The method for preparing a porous moist artificial skin according to claim 5, characterized in that: The freeze-dried film is immersed in the gel solution for 5 to 10 minutes.

10. The use of a porous moist artificial skin as claimed in claim 1, characterized in that: The porous moist artificial skin is used as a wound dressing when treating wounds.

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