A preparation method of a silk fibroin / collagen composite sponge for chronic wound repair

Abstract

The application discloses a preparation method of a silk fibroin / collagen composite sponge for chronic wound repair, and comprises the following steps: S1, a silk fibroin solution with a mass concentration of 0.02-0.07 g / mL is freeze-dried to obtain a silk fibroin sponge; S2, the silk fibroin sponge is immersed in an alcohol solution for incubation, and then is washed and dried to obtain an insoluble silk fibroin sponge; and S3, the insoluble silk fibroin sponge is immersed in a collagen solution with a mass concentration of 0.001-0.006 g / mL and a pH of 5.0-5.5 for soaking, the insoluble silk fibroin sponge absorbs the collagen solution, and then is freeze-dried to obtain the silk fibroin / collagen composite sponge. The silk fibroin / collagen composite sponge prepared by the method has excellent water retention, moisture permeability and water retention capacity, can effectively remove wound exudate, regulate and maintain a moist healing environment, can actively induce tissue regeneration, and can promote wound healing, and can meet the use requirement of ideal wound dressing materials for chronic wounds.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a method for preparing a silk fibroin / collagen composite sponge for chronic wound repair. Background Technology

[0002] Chronic wounds (such as diabetic foot ulcers, pressure injuries, and venous ulcers) are common and challenging clinical problems. A typical symptom is persistent and copious exudation, with a complex healing process and a long treatment period. After wound injury, dressings are needed to immediately seal the wound to prevent excessive loss of body fluids and proteins and to maintain a certain level of moisture to promote the growth of new tissue, while preventing excessive wound fluid from causing infection. Therefore, dressings need to have good water retention, breathability, and moisture retention to provide a suitable balanced humidity environment for the wound. Materials with good water retention can absorb a large amount of wound exudate to keep the wound clean; while a suitable moisture permeability can maintain the moisture balance of the wound, avoid excessive exudate accumulation, and maintain a suitable moist environment to facilitate the transport of nutrients and the transmission of cell signals, promoting cell growth and proliferation, thereby accelerating wound healing. In the middle and late stages of wound healing, when wound exudate has been controlled, the wound begins to dry out, which requires dressings with a relatively slow water evaporation rate (i.e., good moisture retention) to maintain a balanced moist environment for a longer period of time.

[0003] Currently, the routine clinical treatments for chronic wounds include debridement, traditional gauze dressings, negative pressure wound therapy, and moist dressings. While debridement, as a fundamental method of wound management, effectively removes necrotic tissue and biofilm, it often causes severe pain to patients during the procedure. Repeated debridement can also cause secondary damage to newly formed granulation tissue, leading to poor patient compliance. Traditional gauze dressings are dry and tend to adhere tightly to the wound after absorbing exudate. Changing dressings can easily cause mechanical tearing of newly formed epithelial and granulation tissue, resulting in further damage, bleeding, and pain, and increasing the risk of infection. Negative pressure wound therapy effectively promotes drainage, reduces tissue edema, and stimulates granulation tissue growth, achieving good results in the treatment of complex wounds, but its equipment is expensive and treatment costs are high. Moist dressings (such as hydrogels) are a new type of dressing that provides a moist healing environment for the wound, reducing pain during dressing changes to some extent. However, their mechanical strength is generally poor, lacking sufficient mechanical support, making them prone to collapse. They also have limited exudate absorption capacity and may cause maceration of the surrounding skin, thus their use remains quite limited.

[0004] In recent years, biomaterials based on natural proteins have provided new strategies for wound repair. Among them, silk fibroin has attracted much attention due to its excellent biocompatibility and tunable degradation rate. Collagen, as a major component of the extracellular matrix, has outstanding cell affinity and healing-promoting ability. Combining the two is expected to produce ideal dressings with complementary properties. Currently, some studies have attempted to develop silk fibroin-collagen composite scaffolds. For example, Chinese patent application CN107812239A discloses the preparation of a tussah silk fibroin-collagen composite scaffold by directly mixing collagen solution and tussah silk fibroin solution and then freeze-drying it, which shows good water absorption and moisturizing properties; however, actual research has found that the porous structure formed by this composite scaffold is too dense and the pores have poor permeability. Although it has a certain water retention capacity, it seriously hinders the effective permeability of water vapor, resulting in poor moisture permeability. For chronic wounds with continuous and large amounts of exudate, it cannot drain excess exudate in time, which can easily lead to excessive exudate accumulation, causing maceration of the perilesional skin, infection, and delaying the healing process.

[0005] Therefore, developing a silk fibroin-collagen composite dressing material that combines excellent moisture absorption and moisturizing properties with high permeability is of great application value in the treatment of chronic wounds. This material can dynamically regulate and maintain the balance of humidity in the wound microenvironment, promote wound healing, and is capable of dynamically regulating and maintaining the balance of humidity in the wound microenvironment. Summary of the Invention

[0006] To overcome the shortcomings of the existing technology, the present invention aims to provide a method for preparing a silk fibroin / collagen composite sponge for chronic wound repair.

[0007] This invention is achieved through the following technical solution:

[0008] In a first aspect, the present invention provides a method for preparing a silk fibroin / collagen composite sponge for chronic wound repair, comprising the following steps:

[0009] S1. A silk fibroin solution with a mass concentration of 0.02~0.07 g / mL was freeze-dried to obtain a silk fibroin sponge;

[0010] S2. The silk fibroin sponge is immersed in an alcohol solution for incubation, and then washed and dried to obtain an insoluble silk fibroin sponge;

[0011] S3. Immerse the insoluble silk fibroin sponge in a collagen solution with a mass concentration of 0.001~0.006 g / mL and a pH of 5.0~5.5 to allow the insoluble silk fibroin sponge to absorb the collagen solution, and then freeze-dry it to prepare a silk fibroin / collagen composite sponge.

[0012] Furthermore, in step S1, the freeze-drying process involves pre-freezing at -20 to -80°C for 10 to 12 hours, followed by freeze-drying in a vacuum freeze dryer.

[0013] Further, in step S2, the volume percentage concentration of the alcohol solution is ≥80%. The alcohol solution is selected from either an ethanol solution or a methanol solution.

[0014] Furthermore, when the alcohol solution is ethanol, the incubation time is preferably 24-36 hours; when the alcohol solution is methanol, the incubation time is preferably 0.5-2 hours.

[0015] Further, in step S1, the silk fibroin solution is prepared by dissolving soluble silk fibroin in purified water. The soluble silk fibroin of the present invention can be obtained commercially or prepared in-house using existing silk fibroin preparation methods.

[0016] Further, in step S2, the washing is performed with purified water to remove residual alcohol from the sponge.

[0017] Further, in step S2, the drying process includes oven drying, air drying, or freeze drying. Drying conditions can be oven drying at 40-55℃.

[0018] Further, in step S3, the collagen solution is prepared by dissolving collagen in an acetic acid solution with a molar concentration of 0.4-0.6 mol / L or a hydrochloric acid solution with a molar concentration of 14-16 mmol / L, and then adjusting the pH to the required level with a sodium hydroxide solution with a mass concentration of 0.001-0.005 g / mL. Preferably, the collagen is derived from animals such as cattle or pigs. The collagen of this invention can be obtained commercially or prepared in-house using existing collagen preparation methods.

[0019] Furthermore, in step S3, the freeze-drying process involves pre-freezing at -20 to -80°C for 8 to 10 hours, followed by freeze-drying in a vacuum freeze dryer.

[0020] Secondly, the present invention also provides a silk fibroin / collagen composite sponge, which is prepared by the preparation method of the present invention.

[0021] Thirdly, the present invention also provides the application of the aforementioned silk fibroin / collagen composite sponge, which can be used to prepare wound dressings.

[0022] The present invention has the following beneficial effects:

[0023] This invention involves a special composite of silk fibroin and collagen. An insoluble silk fibroin sponge is prepared by freeze-drying a silk fibroin solution and then incubating it in an alcohol solution. This sponge is then immersed in a collagen solution and freeze-dried a second time to obtain a silk fibroin / collagen composite sponge. This composite sponge exhibits excellent water retention, moisture permeability, and water-holding capacity. It effectively absorbs wound exudate and dynamically regulates and maintains a balanced, moist healing environment, preventing excessive exudate accumulation. It promotes fibroblast migration, angiogenesis, and collagen deposition, thereby actively inducing tissue regeneration and promoting wound healing. This represents a shift from "passive covering" to "active repair," meeting the needs of chronic wounds for ideal wound dressing materials. Attached Figure Description

[0024] Figure 1 This is a process flow diagram of the preparation of a silk fibroin / collagen composite sponge for chronic wound repair according to the present invention;

[0025] Figure 2 The results of water evaporation rate tests are for the examples and comparative examples. Detailed Implementation

[0026] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0027] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0028] The following describes some of the materials used in the embodiments and comparative examples of the present invention, but are not limited to these materials:

[0029] Soluble silk fibroin: Hunan Aijian Biotechnology Co., Ltd.

[0030] Collagen: Zhejiang Chongshan Biological Products Co., Ltd.

[0031] Ethanol solution: Anhui Ante Food Co., Ltd.

[0032] Methanol solution: Shanghai Lingfeng Chemical Reagent Co., Ltd.

[0033] Example 1

[0034] A silk fibroin / collagen composite sponge for chronic wound repair is prepared as follows:

[0035] S1. Dissolve soluble silk fibroin in purified water to prepare a silk fibroin solution with a mass concentration of 0.05 g / mL; pre-freeze the silk fibroin solution at -80℃ for 10 h, and then freeze-dry it in a vacuum freeze dryer to obtain silk fibroin sponge;

[0036] S2. The silk fibroin sponge was immersed in an 80% ethanol solution and incubated for 24 hours. Then, the sponge was washed with purified water to remove the ethanol and dried in an oven at 50°C to constant weight to obtain an insoluble silk fibroin sponge.

[0037] S3. Collagen was prepared into a collagen solution with a mass concentration of 0.002 g / mL using 0.5 mol / L acetic acid solution, and the pH was adjusted to 5.3 using sodium hydroxide solution with a mass concentration of 0.002 g / mL. The insoluble silk fibroin sponge obtained in step S2 was immersed in the collagen solution for 1 hour to allow the insoluble silk fibroin sponge to fully absorb the collagen solution. After that, it was taken out and pre-frozen at -80℃ for 8 hours. Then, it was placed in a vacuum freeze dryer for freeze drying to prepare a silk fibroin / collagen composite sponge.

[0038] Example 2

[0039] A silk fibroin / collagen composite sponge for chronic wound repair is prepared as follows:

[0040] S1. Dissolve soluble silk fibroin in purified water to prepare a silk fibroin solution with a mass concentration of 0.05 g / mL; pre-freeze the silk fibroin solution at -80℃ for 10 h, and then freeze-dry it in a vacuum freeze dryer to obtain silk fibroin sponge;

[0041] S2. Immerse the silk fibroin sponge in a methanol solution with a volume percentage concentration of 80% and incubate for 0.5 h. Then wash the sponge with purified water to remove the methanol and dry it in an oven at 50 °C until constant weight to obtain an insoluble silk fibroin sponge.

[0042] S3. Collagen was prepared into a collagen solution with a mass concentration of 0.002 g / mL using 0.5 mol / L acetic acid solution, and the pH was adjusted to 5.3 using sodium hydroxide solution with a mass concentration of 0.002 g / mL. The insoluble silk fibroin sponge obtained in step S2 was immersed in the collagen solution for 1 hour to allow the insoluble silk fibroin sponge to fully absorb the collagen solution. After that, it was taken out and pre-frozen at -80℃ for 8 hours. Then, it was placed in a vacuum freeze dryer for freeze drying to prepare a silk fibroin / collagen composite sponge.

[0043] Example 3

[0044] A silk fibroin / collagen composite sponge for chronic wound repair is prepared as follows:

[0045] S1. Dissolve soluble silk fibroin in purified water to prepare a silk fibroin solution with a mass concentration of 0.07 g / mL; pre-freeze the silk fibroin solution at -80℃ for 10 h, and then freeze-dry it in a vacuum freeze dryer to obtain silk fibroin sponge;

[0046] S2. The silk fibroin sponge was immersed in an 80% ethanol solution and incubated for 36 hours. Then, the sponge was washed with purified water to remove the ethanol and dried in an oven at 40°C to constant weight to obtain an insoluble silk fibroin sponge.

[0047] S3. Collagen was prepared into a collagen solution with a mass concentration of 0.005 g / mL using 0.5 mol / L acetic acid solution, and the pH was adjusted to 5.5 using sodium hydroxide solution with a mass concentration of 0.002 g / mL. The insoluble silk fibroin sponge obtained in step S2 was immersed in the collagen solution for 1 hour to allow the insoluble silk fibroin sponge to fully absorb the collagen solution. After that, it was taken out and pre-frozen at -80℃ for 8 hours. Then, it was freeze-dried in a vacuum freeze dryer to obtain a silk fibroin / collagen composite sponge.

[0048] Comparative Example 1

[0049] An insoluble silk fibroin sponge, the preparation method of which differs from that of Example 1 in that it is not combined with collagen, and the preparation steps are as follows:

[0050] S1. Dissolve soluble silk fibroin in purified water to prepare a silk fibroin solution with a mass concentration of 0.05 g / mL; pre-freeze the silk fibroin solution at -80℃ for 10 h, and then freeze-dry it in a vacuum freeze dryer to obtain silk fibroin sponge;

[0051] S2. Immerse the silk fibroin sponge in an ethanol solution for 24 hours, then wash the sponge with purified water to remove the ethanol, and dry it in an oven at 50°C to constant weight to obtain an insoluble silk fibroin sponge.

[0052] Comparative Example 2

[0053] A silk fibroin / collagen composite sponge is prepared in a manner different from that in Example 1, where the silk fibroin solution and collagen solution are first mixed and then freeze-dried to prepare the silk fibroin / collagen composite sponge. The preparation steps are as follows:

[0054] S1. Dissolve soluble silk fibroin in purified water to prepare a silk fibroin solution with a mass concentration of 0.05 g / mL;

[0055] S2. Prepare a collagen solution with a mass concentration of 0.002 g / mL using 0.5 mol / L acetic acid solution, and adjust the pH to 5.3 using sodium hydroxide solution with a mass concentration of 0.002 g / mL.

[0056] S3. After mixing the silk fibroin solution and collagen solution at a volume ratio of 1:1, pre-freeze at -80℃ for 10h, then freeze-dry in a vacuum freeze dryer, then immerse in an ethanol solution for 24h, wash the sponge with purified water to remove ethanol, and dry in an oven at 50℃ to constant weight to obtain the silk fibroin / collagen composite sponge.

[0057] Comparative Example 3

[0058] A silk fibroin / collagen composite sponge is prepared in a manner that differs from that of Example 1 only in that the pH of the collagen solution in step S3 is 7.5, while the remaining steps are the same as in Example 1.

[0059] Performance characterization:

[0060] 1. Moisture permeability and water retention rate

[0061] The moisture permeability of the samples was determined according to the evaporation method in ASTM E96, "Standard Test Method for Moisture Permeability of Materials," and the Chinese national standard GB / T 12704-1991, "Determination of Moisture Permeability of Fabrics - Permeability Cup Method." The moisture permeability of chronic wound dressings was 83.3–104.2 g / (m³). 2 h) is the most suitable.

[0062] The sample (M0) was soaked in distilled water for 24 hours to ensure it was fully saturated. Water droplets adhering to the surface were then absorbed with filter paper, and the sample was weighed and recorded as M. 终 Calculate the water holding capacity of the sample. The calculation formula is as follows:

[0063] Water holding capacity = (M 终 -M0)×100% / M0

[0064] Table 1. Moisture permeability and water retention of sponges prepared in the examples and comparative examples.

[0065]

[0066] This invention produces a silk fibroin / collagen composite sponge by combining silk fibroin and collagen in a special manner, with a moisture permeability of 98-104 g / (m²). 2 h), and has a water holding capacity of ≥900%; it can effectively absorb wound exudate and dynamically regulate and maintain the balance of a moist healing environment, especially meeting the requirements of chronic wound dressing materials.

[0067] Comparative Example 1, consisting of a single silk fibroin sponge, exhibited reduced water retention and a moisture permeability of only approximately 80 g / (m³). 2 h), when used as a dressing for chronic wounds, it is prone to excessive exudate accumulation, which can lead to infection and delay the healing process.

[0068] Comparative Example 2: Silk fibroin solution and collagen solution were mixed and then freeze-dried to prepare silk fibroin / collagen composite sponge. The resulting porous structure was too dense and the pores had poor permeability, resulting in reduced moisture permeability and water retention.

[0069] In Comparative Example 3, collagen may slightly precipitate in an environment of pH 7.5, which prevents it from being well adsorbed into the silk fibroin sponge and forming a more effective network structure, resulting in a decrease in both moisture permeability and water retention.

[0070] 2. Water evaporation rate

[0071] The sample (M0) was soaked in distilled water for 24 hours to ensure it was fully saturated. Water droplets adhering to the surface were then absorbed with filter paper, and the sample was weighed and recorded as M. 终 The sample was placed in an environment with a temperature of 37℃ and a relative humidity of 39%, and weighed every 5 minutes, recorded as M. n The water evaporation rate of the sample is calculated until the final weight no longer changes, and the results are as follows: Figure 2 As shown. The formula for calculating water evaporation rate is as follows:

[0072] Water evaporation rate = (M 终 -M n )×100% / (M 终 -M0)

[0073] Examples 1 to 3 exhibit a relatively slow water evaporation rate, presumably because both silk fibroin and collagen contain a large number of hydrophilic amino acids. These groups can form hydrogen bonds with water molecules, effectively binding and locking in moisture. The synergistic effect of the two effectively improves the sponge's water retention capacity. Furthermore, the combination of silk fibroin and collagen forms a more complex three-dimensional porous structure. The synergistic effect of the two enables the sponge to form a stable hydration network, effectively preventing moisture loss and providing a long-lasting moisturizing effect.

[0074] Comparative Example 1 is a single silk fibroin sponge with a simple network structure, low porosity, and poor water retention capacity.

[0075] Comparative Example 2: Silk fibroin solution and collagen solution were mixed and then freeze-dried to prepare silk fibroin / collagen composite sponge. During incubation in ethanol solution, ethanol denatured some collagen, destroying the network structure of the composite sponge, resulting in poor water retention capacity and rapid water evaporation rate.

[0076] In Comparative Example 3, collagen may slightly precipitate in an environment of pH 7.5, which prevents it from being well adsorbed into the silk fibroin sponge, thus failing to form an effective network structure and resulting in poor water retention.

[0077] 3. Ethanol residue

[0078] Ethanol residue was determined using gas chromatography. The chromatographic conditions were as follows: column temperature: 60℃, held for 2 min; vaporization chamber temperature: 200℃; detection chamber temperature: 250℃. The results are shown in Table 2.

[0079] Table 2. Ethanol residue in sponges prepared in the examples and comparative examples.

[0080]

[0081] The silk fibroin / collagen composite sponge of the present invention has low ethanol residue, high safety, and is suitable for chronic wound dressing.

[0082] The above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing a silk fibroin / collagen composite sponge for chronic wound repair, characterized by, Includes the following steps: S1. A silk fibroin solution with a mass concentration of 0.02~0.07 g / mL was freeze-dried to obtain a silk fibroin sponge; S2. Immerse the silk fibroin sponge in an ethanol solution with a volume percentage concentration of ≥80% and incubate for 24~36h, then wash and dry to obtain an insoluble silk fibroin sponge; S3. Immerse the insoluble silk fibroin sponge in a collagen solution with a mass concentration of 0.001~0.006 g / mL and a pH of 5.0~5.

5. After the insoluble silk fibroin sponge absorbs the collagen solution, pre-freeze it at -80℃~-20℃ for 8~10 h, and then freeze-dry it in a vacuum freeze dryer to prepare a silk fibroin / collagen composite sponge. In step S1, the silk fibroin solution is prepared by dissolving soluble silk fibroin in purified water.

2. The production method according to claim 1, characterized by, In step S1, the freeze-drying process involves pre-freezing at -80℃ to -20℃ for 10 to 12 hours, followed by freeze-drying in a vacuum freeze dryer.

3. The preparation method according to claim 1, characterized in that, In step S2, the washing is done with purified water; the drying is done by baking, air drying, or freeze drying.

4. The method of claim 1, wherein, In step S3, the collagen solution is prepared by using acetic acid solution with a molar concentration of 0.4~0.6 mol / L or hydrochloric acid solution with a molar concentration of 14~16 mmol / L, and then adjusting the pH to the required level with sodium hydroxide solution with a mass concentration of 0.001~0.005 g / mL.

5. A silk fibroin / collagen composite sponge, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 4.

6. The application of the silk fibroin / collagen composite sponge according to claim 5, characterized in that, Used to prepare wound dressings.

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