Method for the preparation of a biological dressing using a combination of enzymes and use thereof

Abstract

This invention provides a method and application for preparing biological dressings using a combination of enzymes. The preparation method includes static water thawing; dehairing and degreasing; cutting and segmenting; fine trimming and perforation; iodine immersion treatment; purified water immersion treatment; trypsin and neutral protease solution immersion treatment; purified water immersion treatment; antibacterial coating treatment; packaging; and irradiation. This invention significantly improves the antigen removal rate through the combined use of two enzymes. The combined use of these two enzymes enables comprehensive and efficient degradation of different types of heterogeneous proteins, forming a complementary enzymatic process. The combined use of two enzymes can greatly increase the antigen removal rate, significantly improve the biocompatibility of the biological dressing, reduce the risk of immune rejection in clinical applications, and the prepared biological dressing has good biocompatibility and suitable clinical applicability.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, and in particular to a method and application for preparing bio-dressings using a combination of enzymes. Background Technology

[0002] Currently, wound dressings used in clinical practice are mainly divided into three categories: traditional dressings, synthetic dressings, and natural biological dressings. Among them, biological skin-based dressings have unique advantages in wound repair due to their physiological characteristics similar to human skin. They are classified as Class III medical devices and are mainly used for covering and repairing burns and deep wounds.

[0003] The use of enzymes has certain advantages in the preparation of biological dressings, but traditional enzymes are no longer suitable for improving the performance of current biological dressings. Therefore, developing a biological skin dressing that is low in antigenicity, has excellent biocompatibility, stable mechanical properties, can adapt to the needs of different stages of wound healing, and has a simple preparation process and is easy to mass-produce has become an urgent technical problem to be solved in the field of wound repair. Summary of the Invention

[0004] The technical problem to be solved by the present invention is that existing biological dressings have poor biocompatibility and compatibility. Therefore, the present invention provides a method and application for preparing biological dressings by combining enzymes.

[0005] To address the aforementioned technical problems, this invention provides a method for preparing a biological dressing using a combination of enzymes, the preparation method comprising the following steps: (1) Soak the bio-skin in water for the first time to obtain semi-finished product A; (2) Scrape the semi-finished product A obtained in step (1) to obtain semi-finished product B; (3) Cut the semi-finished product B obtained in step (2) to obtain semi-finished product C; (4) Punch holes in the semi-finished product C obtained in step (3) to obtain the semi-finished product D; (5) Soak the semi-finished product D obtained in step (4) in iodine solution to obtain semi-finished product E; (6) Soak the semi-finished product E obtained in step (5) in water for a second time to obtain the semi-finished product F; (7) Soak the semi-finished product F from step (6) in a solution of trypsin and neutral protease to obtain semi-finished product G; (8) Soak the semi-finished product G from step (7) in water for the third time to obtain the semi-finished product H; (9) The semi-finished product H obtained in step (8) is subjected to antibacterial treatment with an antibacterial agent solution to obtain semi-finished product I; (10) Use Co to process the semi-finished product I obtained in step (9). 60Irradiation was performed to obtain the biological dressing.

[0006] The biological dressing product prepared by this invention can purify, clean, and protect wounds, promote granulation tissue growth, cover granulation wounds, chronic ulcers, and pressure sores, and shorten healing time and improve healing effect.

[0007] Preferably, the bio-skin in step (1) includes pigskin or cowhide.

[0008] This invention preferably uses pigskin. The pigskin selected for this invention can be that of Landrace pigs with a growth period of 4 to 6 months, and the surface area of ​​the whole pigskin is greater than 80 square centimeters, excluding the head, tail, belly and limbs.

[0009] The operation in step (1) is mainly the process of thawing in still water.

[0010] Preferably, the soaking time in step (1) is 5 to 9 hours, for example, 5 hours, 7 hours or 9 hours.

[0011] Preferably, the soaking temperature in step (1) is 15~20℃, for example, it can be 15℃, 16℃, 17℃, 18℃, 19℃ or 20℃.

[0012] Preferably, the mass ratio of the bio-skin to water in step (1) is 1:(5~10), for example, it can be 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10, etc.

[0013] Step (2) mainly involves the process of removing hair and grease. Lay the semi-finished product A flat on the workbench with the hair side facing up. Use a sharp knife to scrape against the direction of hair at a 45-degree angle with appropriate force until no hair is visible on the skin. Then turn it over and use a blunt knife to scrape against the direction of hair at a 45-degree angle with appropriate force until no fat is visible to the naked eye, thus obtaining the semi-finished product B.

[0014] Step (3) involves the cutting and segmentation process. The semi-finished product B is laid flat on the operating table and vertically cut into appropriate sizes. The specific sizes are determined according to the specifications of the subsequent processing equipment and product requirements. After cleaning the segmented heterologous hides, they are fixed on a drum-type leather cutter for longitudinal cutting. The cutting thickness is controlled by adjusting the blade gap of the leather cutter to obtain the semi-finished product C.

[0015] In step (4), the process of fine-tuning and drilling is to lay the semi-finished product C flat on the worktable, use a special hand-held bone-scraping knife to first scrape off the excess fat, stray hairs and micro-hairs of the dermis, and then scrape off the stratum corneum of the epidermis. Adjust the force according to the thickness of the skin piece, and fine-tune it to the thickness and size required by the product. After trimming, drill holes at a certain interval and hole diameter until the semi-finished product D is obtained.

[0016] Preferably, the concentration of the iodine solution in step (5) is 100~150 mg / L, for example, it can be 100 mg / L, 120 mg / L, 130 mg / L, 150 mg / L, etc.

[0017] Preferably, in step (5), each square centimeter of semi-finished product D is soaked in 1~3 mL of povidone-iodine solution.

[0018] Preferably, the soaking time is 5 to 15 minutes, for example, 5 minutes, 8 minutes, 10 minutes, 12 minutes or 15 minutes.

[0019] Preferably, in step (6), 1 to 3 mL of water is used per square centimeter of semi-finished product E, for example, 1 mL, 2 mL, 3 mL, etc.

[0020] Preferably, in step (6), after soaking, the water is shaken, the water is changed, and the shaking and water changing are repeated until the pH value of the water is 5.5~6.5, for example, it can be 5.5, 5.8, 6.2 or 6.5, etc.

[0021] In this invention, the pH of the purified water is stabilized at 5.5~6.5 after replacement (close to the physiological pH range of human skin), which can avoid the damage of the core structure of the biological skin matrix such as collagen fibers and elastic fibers by excessively acidic or alkaline environments, and prevent the denaturation and inactivation of bioactive components.

[0022] Preferably, the mass ratio of trypsin to neutral protease in step (7) is (1~3):1, for example, it can be 1:1, 2:1, 3:1, etc.

[0023] The neutral protease selected in this invention (e.g., Bacillus subtilis neutral protease with an enzyme activity of 10,000-20,000 U / g) can efficiently degrade insoluble impurities and improve the antigen removal effect. Neutral protease can specifically act on insoluble fibrin, elastin, and residual cell debris in the bio-dress, degrading them into water-soluble small peptides. This effectively removes foreign antigens (such as cell surface antigens and structural protein antigens) from the bio-dress, preventing immune rejection after subsequent implantation into the human body. This is a core prerequisite for the biocompatibility of bio-dresses. Compared to the limitation of trypsin alone, which can only degrade soluble proteins, neutral protease can specifically address insoluble impurities. It has a mild enzymatic hydrolysis effect, which can protect the integrity of collagen structure: neutral protease acts in a neutral pH environment (7.0~7.5) and can achieve efficient enzymatic hydrolysis without the assistance of strong alkali. Compared with the corrosive and degradative effects of alkali metal hydroxides on biological skin collagen fibers, it can preserve the natural three-dimensional network structure of collagen fibers and the key to cell compatibility to the greatest extent. It can provide a stable scaffold for wound healing, promote epithelial cell attachment and proliferation, and make the prepared biological dressing more skin-friendly.

[0024] Furthermore, this invention employs a dual-enzyme system combining trypsin and neutral protease. The two enzymes work synergistically to significantly improve the antigen removal rate. Trypsin focuses on degrading soluble proteins and intracellular soluble antigens in the biological dressing, while neutral protease focuses on degrading insoluble proteins and fibrous antigens. The combined use of these two enzymes achieves comprehensive and efficient degradation of various types of proteins, forming complementary enzymatic reactions. The combined use of these two enzymes significantly increases the antigen removal rate, greatly improving the biocompatibility of the biological dressing and reducing the risk of immune rejection in clinical applications.

[0025] Furthermore, both enzymes function under neutral pH (7.0-7.5) and mild temperature conditions, eliminating the need for strong alkalis and avoiding damage to the collagen structure. The synergistic effect of the two enzymes shortens the hydrolysis time (ideal results can be achieved in 2-4 hours), significantly improving production efficiency compared to the 6-8 hours required for single-enzyme hydrolysis. Most importantly, the combined use of the two enzymes optimizes the surface properties of the biological dressing, promoting wound healing. Synergistic hydrolysis creates uniform micropores on the surface of the biological dressing, ensuring breathability and enhancing its absorption capacity for wound exudate. Simultaneously, the increased number of residual collagen surfactant groups after hydrolysis promotes the attachment and proliferation of epithelial cells and fibroblasts on the wound surface, accelerating wound healing. Compared to dressings prepared using existing technologies, the healing cycle can be shortened by 1-2 days, improving clinical application outcomes.

[0026] Preferably, in step (7), each square centimeter of semi-finished product F is soaked in a solution of 1 to 3 mL (e.g., 1 mL, 2 mL, or 3 mL, etc.); Preferably, the soaking time is 2 to 4 hours, for example, 2 hours, 3 hours or 4 hours.

[0027] Preferably, the antibacterial agent in step (9) is zinc sulfadiazine ointment.

[0028] Preferably, the mass concentration of the antibacterial agent solution in step (9) is 2.5~4 g / L, for example, it can be 2.5 g / L, 3 g / L or 4 g / L.

[0029] On the other hand, the present invention provides a biological dressing prepared by the preparation method described above.

[0030] This invention utilizes a combination of physical, chemical, and biological methods to sterilize and reduce antigens in biological skin, preserving the dermal and epidermal structures, resulting in clinical efficacy superior to freeze-dried xenograft skin.

[0031] The present invention also provides the application of a biological dressing in the preparation of a wound covering material.

[0032] The wound surface of the present invention can be a wound surface formed by burns, trauma, etc.

[0033] Implementing this invention has the following beneficial effects: This invention significantly improves the antigen removal rate through the combined use of two enzymes. The combined use of these two enzymes enables comprehensive and efficient degradation of various heterogeneous proteins, forming a complementary enzymatic process. The combined use of the two enzymes greatly enhances the antigen removal rate, significantly improving the biocompatibility of the biological dressing and reducing the risk of immune rejection in clinical applications. The prepared biological dressing exhibits good biocompatibility and suitable clinical applicability. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example 1

[0035] The heterologous skin of this invention is selected from Landrace pigs with a growth period of 4-6 months. Excluding the head, tail, belly and limbs, the size of the whole pigskin is ≥80 square centimeters.

[0036] (1) Thawing in still water. Let it stand in a ratio of 1 kg of pigskin to 7 kg of purified water, and keep the water temperature at around 15 degrees Celsius.

[0037] (2) Dehairing and degreasing. Lay the semi-finished product A flat on the worktable with the hair side facing up. Use a sharp knife to scrape against the grain at a 45-degree angle with appropriate force until no hair is visible on the surface. Then turn it over and use a blunt knife to scrape against the grain at a 45-degree angle with appropriate force until no fat is visible. This yields the semi-finished product. (3) Cutting and segmenting. Lay the semi-finished product B flat on the worktable and cut it vertically to the appropriate size. The specific size depends on the specifications of the subsequent processing equipment and the product requirements. After cleaning the segmented foreign skin, fix it on a drum-type skin cutting machine for longitudinal cutting. Control the cutting thickness by adjusting the gap between the cutting machine blades to obtain the semi-finished product C.

[0038] (4) Fine trimming and drilling. Lay the semi-finished product C flat on the worktable. Use a special hand-held boning knife to first scrape off the excess fat, stray hairs and micro-hairs in the dermis, and then scrape off the stratum corneum. Adjust the force according to the thickness of the skin piece, and fine trim to the required thickness and size of the product. After trimming, drill holes at a certain interval and with a certain diameter until the semi-finished product D is obtained.

[0039] (5) Iodine immersion treatment. Use an iodine solution with a concentration of 120 mg / L. Take the corresponding amount of iodine solution at a ratio of 3 ml per square centimeter of pigskin and immerse for 12 minutes to obtain semi-finished product E.

[0040] (6) Soaking in purified water. Take 2 ml of purified water per square centimeter of semi-finished product E, place semi-finished product E and the purified water in the same container, place it on a shaker and shake for 25 minutes, replace the purified water, and place it on a shaker and shake for 25 minutes again; repeat the above steps until the pH value of the replaced purified water is between 5.5 and 6.5, then discard the purified water to obtain semi-finished product F.

[0041] (7) Soaking treatment with a mixture of trypsin and neutral protease. Prepare a solution of trypsin and neutral protease (Bacillus subtilis neutral protease) at a mass ratio of 2:1 and cool it in an ice bath. Soak the pigskin in the mixture at a ratio of 2 ml per square centimeter for 3 hours to obtain semi-finished product G.

[0042] (8) Soaking in purified water. Same as step (6), to obtain semi-finished product H.

[0043] (9) Antibacterial treatment by brushing. Use 5% zinc sulfadiazine ointment, dilute 1g with 250ml of purified water to make an antibacterial solution. In a clean environment, apply the antibacterial solution evenly to the dermal surface of the pigskin to obtain semi-finished product I.

[0044] (10) Packaging and Irradiation. Semi-finished product I is placed in an inner packaging bag, laid flat inside, and the inner packaging bag is sealed. Finally, it is irradiated with cobalt-Co. 60 Irradiation ultimately yields biological dressings.

[0045] The antigen removal rate of Example 1 was 98.2%, the collagen structure was good, and there was no obvious degradation. Example 2

[0046] The difference between this example and Example 1 is that in step (7), trypsin and neutral protease are prepared in a mass ratio of 1:1.

[0047] The antigen removal rate of Example 2 was 92.5%, the collagen structure was good, and there was no obvious degradation. Example 3

[0048] The difference between this example and Example 1 is that in step (7), trypsin and neutral protease are prepared in a mass ratio of 3:1.

[0049] The antigen removal rate of Example 3 was 97.8%, with good collagen structure and slight degradation.

[0050] Comparative Example 1 The difference between this comparative example and Example 1 is that this comparative example does not include step (5) iodine immersion treatment.

[0051] Compared with Example 1, the resulting product is whiter in appearance, has similar softness, and shows almost no change in antigen removal rate, tensile deformation rate, yield strength, and pH value, but its ultimate strength increases by an average of about 300%.

[0052] Comparative Example 2 The difference between this comparative example and Example 1 is that step (5) in this comparative example does not include trypsin.

[0053] Compared with Example 1, the resulting product had a similar appearance and color, but poorer softness, a slightly alkaline pH, and showed an average increase of approximately 60% in tensile deformation rate and yield strength, and an average increase of approximately 280% in ultimate strength. The antigen removal rate was 64.5%.

[0054] Comparative Example 3 The difference between this comparative example and Example 1 is that step (7) in this comparative example does not include neutral protease.

[0055] Compared with Example 1, the resulting product had a similar appearance and color, but poorer softness, a slightly acidic pH value, and showed an average increase of approximately 17% in tensile deformation rate and yield strength, and an average increase of approximately 100% in ultimate strength. The antigen removal rate was 75.2%.

[0056] Comparative Example 4 The difference between this comparative example and Example 1 is that the neutral protease in this comparative example is replaced with sodium hydroxide solution.

[0057] In this embodiment, the antigen removal rate was 90.1%, the collagen structure was generally normal, and there was local collagen degradation.

[0058] Comparative Example 5 The difference between this comparative example and Example 1 is that this comparative example does not include step (7).

[0059] Compared with Example 1, the resulting product has a yellowish appearance, poor softness, similar pH value, and an average increase of about 40% in tensile deformation rate and yield strength, an average increase of about 330% in ultimate strength, and a deantigenation rate of 2.8%.

[0060] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a biological dressing using a combination of enzymes, characterized in that, The method includes the following steps: (1) Soak the bio-skin in water for the first time to obtain semi-finished product A; (2) Scrape the semi-finished product A obtained in step (1) to obtain semi-finished product B; (3) Cut the semi-finished product B obtained in step (2) to obtain semi-finished product C; (4) Punch holes in the semi-finished product C obtained in step (3) to obtain the semi-finished product D; (5) Soak the semi-finished product D obtained in step (4) in iodine solution to obtain semi-finished product E; (6) Soak the semi-finished product E obtained in step (5) in water for a second time to obtain the semi-finished product F; (7) Soak the semi-finished product F from step (6) in a solution of trypsin and neutral protease to obtain semi-finished product G; (8) Soak the semi-finished product G from step (7) in water for the third time to obtain the semi-finished product H; (9) The semi-finished product H obtained in step (8) is subjected to antibacterial treatment with an antibacterial agent solution to obtain semi-finished product I; (10) Use Co to process the semi-finished product I obtained in step (9). 60 Irradiation was performed to obtain the biological dressing.

2. The preparation method according to claim 1, characterized in that, The biological hide mentioned in step (1) includes pigskin or cowhide; Preferably, the soaking time in step (1) is 5-9 hours; Preferably, the soaking temperature in step (1) is 15~20℃; Preferably, the mass ratio of the bio-skin to water in step (1) is 1:(5~10).

3. The preparation method according to claim 1, characterized in that, The concentration of the iodine solution in step (5) is 100~150 mg / L.

4. The preparation method according to claim 1, characterized in that, In step (5), each square centimeter of semi-finished product D is soaked in 1~3 mL of povidone-iodine solution; Preferably, the soaking time is 5 to 15 minutes.

5. The preparation method according to claim 1, characterized in that, In step (6), 1~3mL of water is used per square centimeter of semi-finished product E; Preferably, after soaking in step (6), the water is shaken, the water is changed, and the shaking and water changing are repeated until the pH value of the water is 5.5~6.

5.

6. The preparation method according to claim 1, characterized in that, The mass ratio of trypsin to neutral protease in step (7) is (1~3):

1.

7. The preparation method according to claim 1, characterized in that, In step (7), each square centimeter of semi-finished product F is soaked in 1~3 mL of solution; Preferably, the soaking time is 2 to 4 hours.

8. The preparation method according to claim 1, characterized in that, The antibacterial agent in step (9) is zinc sulfadiazine ointment; Preferably, the mass concentration of the antibacterial agent solution in step (9) is 2.5~4 g / L.

9. The biological dressing prepared by the method according to any one of claims 1 to 8.

10. The use of the biological dressing according to claim 9 in the preparation of a wound covering material.