Preparation method of composite dressing for inhibiting scars

Abstract

The invention provides a preparation method of a composite dressing for inhibiting scars. The preparation method comprises the following steps: 1) preparing a CMC (sodium carboxymethyl cellulose) sponge layer; 2) respectively preparing a silk fibroin electrostatic spinning solution and a polylactic acid / retinoic acid electrostatic spinning solution; and 3) electrospinning the silk fibroin electrostatic spinning solution to the surface of the CMC sponge layer, and then electrospinning the polylactic acid / retinoic acid electrostatic spinning solution to the CMC sponge layer to obtain the sponge-nanofiber-nanofiber composite dressing. All the layers of the composite dressing jointly achieve the effects of optimization of a wound microenvironment, one-way management of wound exudate, directional growth of cells and drug regulation and control, and therefore the purposes of accelerating wound healing and reducing abnormal scar formation are achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of dressings, and in particular relates to a method for preparing a composite dressing for inhibiting scars. Background Art

[0002] Wound healing is a complex and continuous physiological process, from the inflammatory phase, proliferation phase to the remodeling phase, each stage has its own unique cellular and molecular events. In the early stage of trauma, inflammatory cells gather rapidly to remove pathogens and tissue debris; then, fibroblasts are activated and proliferate in large numbers, participating in the deposition and reconstruction of the extracellular matrix. However, when local inflammation persists or is too severe, fibroblasts will proliferate abnormally and differentiate into myofibroblasts, leading to unbalanced deposition and disordered arrangement of collagen (mainly type I and type III), thus forming hypertrophic or hypertrophic scars. This scar not only affects the beauty of the skin, but also causes the tissue to lose its normal elasticity and function.

[0003] At present, the commonly used methods for scarless wound treatment include pressure therapy (such as elastic bandages, scar pressure clothing), drug therapy (such as silicone preparations, hormone injections, topical drugs) and surgical intervention. Although these treatment methods are still widely used, they inevitably have some defects. For example, although pressure therapy can reduce capillary supply and inhibit scar hyperplasia through continuous external pressure, and the price is relatively low, it will have problems such as poor air permeability, difficulty in maintaining a moist microenvironment, lack of bioactive signal guidance, and easily lead to local discomfort, skin ulceration or poor compliance. Although drug therapy is relatively simple to use, it may cause local skin atrophy, capillary dilation, pigmentation or depression. Surgical intervention can directly remove scars and re-suture the wound to make the scar smaller, but it is only suitable for linear scars or larger scars, and pressure therapy or laser is still needed after surgery to reduce the recurrence rate. These defects have led to the fact that existing clinical treatment methods are increasingly unable to meet the various requirements of scarless treatment.

[0004] In view of the problems existing in existing scar-free dressings and in order to solve the scar problem caused by abnormal cell behavior, it is necessary to develop a multi-layer structure with rapid fluid conduction capabilities and a multifunctional dressing that promotes fibroblast proliferation by sustained release of related drugs and provides a scaffold for orderly cell growth. Summary of the invention

[0005] In order to solve the problems existing in the prior art such as repeated infection caused by severe wound exudate, unstable structure, poor biocompatibility, etc., the present invention provides a method for preparing a composite dressing for inhibiting scars. The combination of each layer jointly realizes the optimization of the wound microenvironment, the unidirectional management of wound exudate, the directional growth of cells and the drug regulation effect, promotes the proliferation of fibroblasts by sustained-release drugs and provides an effective cell growth scaffold, reduces abnormal scars, and takes into account mechanical properties, high strength, flexibility, etc.

[0006] The present invention solves the technical problem by adopting the following technical solutions:

[0007] The first object of the present invention is to provide a method for preparing a composite dressing for inhibiting scars, comprising the following steps:

[0008] 1) preparing a CMC (sodium carboxymethyl cellulose) sponge layer;

[0009] 2) preparing a silk fibroin electrospinning solution and a polylactic acid / retinoic acid electrospinning solution respectively;

[0010] 3) After the silk fibroin electrospinning solution is electrospun onto the surface of the CMC sponge layer, the polylactic acid / retinoic acid electrospinning solution is sequentially electrospun onto the layer to obtain a sponge-nanofiber-nanofiber composite dressing.

[0011] Furthermore, after the silk fibroin electrospinning is completed, the surface of the dressing is treated with ethanol vapor and then dried.

[0012] Furthermore, the ethanol vapor treatment was performed using 75% ethanol at a temperature of 25° C. for 1 h.

[0013] Furthermore, the preparation method of the CMC sponge layer is: treating cotton fiber with alkali solution and reacting with a carboxymethylating agent, then dissolving the solution with deionized water to obtain a CMC solution, pouring the CMC solution into a mold, and freeze-drying to obtain a highly absorbent CMC sponge.

[0014] Furthermore, the freeze-drying conditions are freezing at -80°C for 24 hours and then freeze-drying for 48 hours.

[0015] Furthermore, the mass fraction of the CMC solution is 2-10%.

[0016] Furthermore, the silk fibroin electrospinning solution is obtained by dissolving silk fibroin in a hexafluoroisopropanol solution.

[0017] Furthermore, the mass fraction of silk fibroin in the silk fibroin electrospinning solution is 4-8%.

[0018] Furthermore, the polylactic acid / retinoic acid electrospinning solution is obtained by adding polylactic acid and retinoic acid in proportion to a hexafluoroisopropanol solution.

[0019] Furthermore, the mass fraction of the polylactic acid is 5-15%, and the mass fraction of the retinoic acid is 0.01-0.5%.

[0020] Furthermore, the electrospinning process parameters of the silk fibroin electrospinning solution are: using a No. 20 spinning needle, a spinning voltage of 15 to 30 kV, a distance of 10 to 25 cm between the spinning needle and the receiver, a solution flow rate of 0.5 to 5 mL / h, and a spinning time of 10 to 60 min.

[0021] Furthermore, the electrospinning process parameters of the polylactic acid / retinoic acid electrospinning solution are: using a No. 20 spinning needle, a spinning voltage of 15 to 30 kV, a distance of 10 to 25 cm between the spinning needle and the receiver, a solution flow rate of 0.5 to 5 mL / h, and a spinning time of 10 to 60 min.

[0022] Furthermore, nanofiber structures with different orientations can be obtained by adjusting the rotation speed of the collector. When the rotation speed of the collector is 20 rpm to 100 rpm, disorderly arranged polylactic acid / retinoic acid nanofibers are obtained.

[0023] Furthermore, nanofiber structures with different orientations can be obtained by adjusting the rotation speed of the collector. When the rotation speed of the collector is 2000-3000 rpm, orderly arranged polylactic acid / retinoic acid nanofibers are obtained.

[0024] The second object of the present invention is to provide a composite dressing obtained by the above method, wherein the dressing comprises a highly absorbent CMC sponge outer layer, a hydrophilic silk fibroin nanofiber membrane, and an ordered polylactic acid / retinoic acid nanofiber inner layer.

[0025] Compared with the prior art, the beneficial technical effects of the present invention are:

[0026] The present invention realizes the optimization of wound microenvironment, unidirectional management of wound exudate, directional cell growth and drug regulation through the highly absorbent CMC (sodium carboxymethyl cellulose) sponge outer layer, hydrophilic silk protein nanofiber membrane and polylactic acid / retinoic acid nanofiber inner layer, thereby achieving the purpose of accelerating wound healing and reducing abnormal scar formation. The orderly arranged nanofibers prepared by electrospinning technology provide a clear growth channel for cells through the "contact guidance" effect, so that fibroblasts and keratinocytes can migrate and proliferate in an orderly manner along the fiber direction. This bionic structure not only provides a scaffold for cell adhesion, which helps the rapid closure of the wound edge, but also effectively inhibits the uneven collagen deposition caused by random cell diffusion, helps to regulate cell migration and tissue remodeling, thereby reducing the risk of irregular collagen deposition and scar formation.

[0027] The present invention loads retinoic acid drugs in the inner layer of ordered nanofibers. As a metabolite of vitamin A, RA (retinoic acid) can regulate intracellular gene expression, activate the Wnt signaling pathway, and promote the secretion of vascular endothelial growth factor, thereby accelerating the proliferation and differentiation of fibroblasts and keratinocytes. In addition, RA can also regulate the synthesis and degradation of ECM, help achieve the balanced deposition of collagen type I and type III, and improve the structure of new tissues. RA is loaded in directional PLA (polylactic acid) nanofibers, and through the slow degradation and controlled release characteristics of the fiber structure, not only its sustained sustained release effect is guaranteed, but also long-term and stable biological stimulation is provided for wound repair. It can regulate the proliferation of fibroblasts and the deposition of collagen in the early stage of wound healing, optimize the tissue repair process, and then reduce abnormal scar formation.

[0028] The present invention integrates three functions: fluid absorption, transition, and cell guidance / drug sustained release. It not only physically realizes the effective management and unidirectional transmission of exudate, but also provides an ECM-like scaffold to promote directional cell growth, while taking into account mechanical strength and flexibility, making the dressing more stable and easy to operate in actual use.

[0029] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, it can be implemented according to the contents of the specification. In order to make the above and other purposes, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are listed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The cross section of the composite dressing of the present invention and the electron microscope scanning image of each layer of the composite dressing are shown in FIG.

[0031] Figure 2 This is a graph showing the water conductivity of the composite dressing of the present invention and a commercially available Janus-type dressing.

[0032] Figure 3 The figure is a comparison chart of the application effects of various dressings of the present invention in the rabbit ear model.

[0033] Figure 4 This is a cytoskeleton staining diagram of the present invention.

[0034] Figure 5 This is a diagram showing the experimental effect of the in vitro exudate simulation device of the present invention. DETAILED DESCRIPTION

[0035] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the following embodiments are only exemplary illustrations and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope of protection that the present invention is intended to protect.

[0036] In addition, unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0037] Embodiment 1:

[0038] A method for preparing a composite dressing for inhibiting scars comprises the following steps:

[0039] 1) Preparation of CMC sponge layer:

[0040] The cotton fiber is treated with alkali solution and then reacted with a carboxymethylating agent to form partially carboxymethylated cotton fiber. The partially carboxymethylated cotton fiber solution is dissolved in deionized water to obtain a 4% CMC solution, which is then poured into a rectangular polytetrafluoroethylene mold, frozen at -80°C for 24 hours, and then freeze-dried for 48 hours to obtain a highly absorbent CMC sponge.

[0041] 2) Preparation of silk fibroin electrospinning solution:

[0042] 1.2 g of silk fibroin was dissolved in 20 g of hexafluoroisopropanol solution to prepare a 6% silk fibroin electrospinning solution, and magnetic stirring was performed for 12 h until the solution was completely dissolved.

[0043] Preparation of polylactic acid / retinoic acid electrospinning solution:

[0044] 1.6 g of polylactic acid and 10 mg of retinoic acid were dissolved in 20 mL of hexafluoroisopropanol to obtain a mixed solution of 8% polylactic acid and 0.05% retinoic acid, and magnetic stirring was performed for 12 h until the solution was completely dissolved.

[0045] 3) Preparation of sponge-nanofiber-nanofiber composite dressing:

[0046] The CMC sponge was fixed on the receiver, and the silk fibroin electrospinning solution was loaded into a 5 mL syringe for electrospinning on the surface of the CMC sponge. The electrospinning parameters were as follows: 20-gauge needle, spinning voltage 25 kV, needle distance to receiver 12 cm, solution flow rate 1 mL / h, and spinning time 30 min.

[0047] After the electrospinning was completed, the obtained sponge-nanofiber dressing film was treated with 75% ethanol vapor at 25 °C for 1 h and then vacuum dried at room temperature for 2 h.

[0048] The obtained sponge-nanofiber dressing was fixed on the receiver, and the polylactic acid / retinoic acid electrospinning solution was loaded into a 5 mL syringe and electrospun on the surface of the dressing nanofiber layer. The electrospinning parameters were as follows: 20-gauge needle, spinning voltage 20 kV, needle distance 20 cm from the receiver, solution flow rate 0.8 mL / h, spinning time 20 min, and collector speed 2150 rpm.

[0049] Embodiment 2:

[0050] A method for preparing a composite dressing for inhibiting scars comprises the following steps:

[0051] 1) Preparation of CMC sponge layer:

[0052] The cotton fiber is treated with alkali solution and then reacted with a carboxymethylating agent to form partially carboxymethylated cotton fiber. The partially carboxymethylated cotton fiber solution is dissolved in deionized water to obtain a 4% CMC solution, which is then poured into a rectangular polytetrafluoroethylene mold, frozen at -80°C for 24 hours, and then freeze-dried for 48 hours to obtain a highly absorbent CMC sponge.

[0053] 2) Preparation of silk fibroin electrospinning solution:

[0054] 1.2 g of silk fibroin was dissolved in 20 g of hexafluoroisopropanol solution to prepare a 6% silk fibroin electrospinning solution, and magnetic stirring was performed for 12 h until the solution was completely dissolved.

[0055] Preparation of polylactic acid / retinoic acid electrospinning solution:

[0056] 1.6 g of polylactic acid and 10 mg of retinoic acid were dissolved in 20 mL of hexafluoroisopropanol to obtain a mixed solution of 8% polylactic acid and 0.05% retinoic acid, and magnetic stirring was performed for 12 h until the solution was completely dissolved.

[0057] 3) Preparation of sponge-nanofiber-nanofiber composite dressing:

[0058] The CMC sponge was fixed on the receiver, and the silk fibroin electrospinning solution was loaded into a 5 mL syringe for electrospinning on the surface of the CMC sponge. The electrospinning parameters were as follows: 20-gauge needle, spinning voltage 25 kV, needle distance to receiver 12 cm, solution flow rate 1 mL / h, and spinning time 30 min.

[0059] After the electrospinning was completed, the obtained sponge-nanofiber dressing film was treated with 75% ethanol vapor at 25 °C for 1 h and then vacuum dried at room temperature for 2 h.

[0060] The obtained sponge-nanofiber dressing was fixed on the receiver, and the polylactic acid / retinoic acid electrospinning solution was loaded into a 5 mL syringe for electrospinning on the surface of the dressing nanofiber layer. The electrospinning parameters were as follows: 20-gauge needle, spinning voltage 20 kV, needle distance 20 cm from the receiver, solution flow rate 0.8 mL / h, spinning time 20 min, and collector speed 50 rpm.

[0061] Comparative Example 1:

[0062] The difference from Example 1 is that the polylactic acid / retinoic acid nanofiber inner layer is replaced by a polylactic acid nanofiber inner layer.

[0063] Preparation of polylactic acid electrospinning solution:

[0064] 1.6 g of polylactic acid was dissolved in 20 mL of hexafluoroisopropanol to obtain a mixed solution of polylactic acid with a concentration of 8%, and the solution was magnetically stirred for 12 h until it was completely dissolved.

[0065] After the sponge-nanofiber dressing was obtained, the sponge-nanofiber dressing was fixed on a receiver, and the polylactic acid electrospinning solution was loaded into a 5 mL syringe for electrospinning on the surface of the dressing nanofiber layer. The electrospinning parameters were as follows: 20-gauge needle, spinning voltage 20 kV, needle distance 20 cm from receiver, solution flow rate 0.8 mL / h, spinning time 20 min, and collector speed 2150 rpm.

[0066] Example 3: Composite dressing performance test

[0067] 1. Organizational structure

[0068] Each layer of the composite dressing obtained in Example 1 was scanned by electron microscope, and the polylactic acid / retinoic acid nanofiber layer (R-PLA / RA) of the composite dressing obtained in Example 2 was scanned by electron microscope. The cross-section of the polylactic acid / retinoic acid nanofiber layer (A-PLA / RA) of Example 1 was scanned by electron microscope after it was cut open.

[0069] See attached Figure 1 It can be seen that each layer of the composite dressing of the present invention is successfully constructed, each layer has different structural characteristics, and each layer presents a clear layered structure and is combined with each other. When the polylactic acid / retinoic acid nanofiber layer is electrospun, when the collector speed is 50rpm, the nanofibers are randomly arranged without rules; when the collector speed is 2000-3000rpm, the nanofibers are arranged asymmetrically in the same direction.

[0070] 2. Liquid absorption and permeability test of composite dressing

[0071] The composite dressing of Example 1 of the present invention and the commercially available Janus-type dressing were respectively covered on the liquid container, and the container was allowed to continuously flow liquid upward, fluorescent material was added to the liquid, and the liquid absorption on the cross section of the dressing was continuously observed. Another piece of the CMC-SF-PLA / RA composite dressing of the present application was taken, and the liquid with the fluorescent material added was slowly dripped into the dressing with a straw, and the cross section of the dressing was observed.

[0072] See attached Figure 2 It can be seen that the composite dressing of the present invention and the Janus dressing have obvious upward liquid conduction capabilities at the 3rd second, and as time goes by, the liquid continues to be pumped upward, so that the liquid coverage area gradually increases until the liquid is pumped to the upper surface of the dressing. Compared with the Janus dressing, the CMC-SF-PLA / RA composite dressing of the present application has better liquid conduction capabilities and faster speed, pumping the liquid to the dressing surface in 9 seconds. The composite dressing of the present invention also has good permeability and can quickly penetrate and wet the entire dressing.

[0073] 3. Scar elimination performance test

[0074] A wound of the same size was formed on the rabbit ear, and the composite dressing sample obtained in Example 1 of the present invention, the composite dressing obtained in Comparative Example 1, and the commercially available Si-FD dressing were respectively used for pasting to promote healing, and the dressings were changed regularly every day. At the same time, a control example was set up, in which a scar of the same size was formed on the rabbit ear, and after disinfection, it was covered with ordinary gauze, and the gauze was changed regularly every day.

[0075] See attached Figure 3 It can be seen that the wound healing and skin cell growth of the composite dressings of Example 1 of the present invention and Comparative Example 1 proceed simultaneously. While the wound is healing, the skin cells at the wound are regenerating. On the 28th day, the wound treated by Example 1 of the present invention has been basically healed, with only a very small scar that has not been eliminated. The skin tissue around the scar has regrown and is close to the original skin tissue. The dressing of Comparative Example 1 does not add retinoic acid. Although there are significant improvements in hemostasis and wound healing, the skin tissue growth ability is insufficient. Although the skin begins to grow after 28 days, the scar does not disappear. The Si-FD dressing has a strong ability to promote wound healing. The wound area has been greatly reduced on the 14th day, but the Si-FD dressing does not have the ability to promote skin tissue growth. On the 28th day, the wound has been completely healed, but the scar has not been reduced.

[0076] 4. Relationship between nanofiber arrangement and cell growth

[0077] The polylactic acid / retinoic acid nanofiber layer of the composite dressing obtained in Example 1 and Example 2 of the present invention was cut into the required size, sterilized and placed in a culture plate, cells in the logarithmic growth phase were digested, resuspended in a serum-containing culture medium, added to the culture plate, and cultured so that they were evenly distributed. After culturing for a certain period of time, the culture solution was aspirated, the cells were fixed, and then stained with FITC, DAPI, and Merge dyes respectively after cell permeabilization.

[0078] See attached Figure 4 It can be seen that compared with the polylactic acid / retinoic acid nanofiber layer (R-PLA / RA) in Example 2, the angle between the attached cells and the nanofibers in the polylactic acid / retinoic acid nanofiber layer (A-PLA / RA) in Example 1 is within ±15°, and the parallel arranged nanofibers are more effective in guiding the directional growth of cells.

[0079] 5. Liquid conduction ability experiment

[0080] Fresh pig skin was used to simulate skin for in vitro simulation experiments. A large amount of water was injected into the surface of the pig skin with a needle, and then dressings were applied, using the composite dressing of Example 1 of the present invention and the commercially available Si-FD dressing, respectively. After 60 minutes, the dressings were removed for observation.

[0081] See attached Figure 5 After 60 minutes, there was a large amount of liquid on the surface of the pig skin pasted with the Si-FD dressing, and the pig skin surface was moist; while the pig skin surface pasted with the composite dressing of the present application was dry, without any liquid residue. It can be seen that the composite dressing has good liquid conduction ability, which can quickly conduct the liquid exuded from the skin surface to the surface, so that the skin surface maintains a moist but not wet microenvironment; while the commercial dressing has poor liquid conduction ability, which keeps the wound in a moist environment for a long time, which is not conducive to wound healing.

[0082] The serial numbers of the above embodiments of the present invention are only for description and do not represent the advantages or disadvantages of the embodiments.

[0083] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

1. A method for preparing a composite dressing for inhibiting scars, characterized in that: The following steps are involved: 1) preparing a CMC sponge layer; 2) preparing a silk fibroin electrospinning solution and a polylactic acid / retinoic acid electrospinning solution respectively; 3) After the silk fibroin electrospinning solution is electrospun onto the surface of the CMC sponge layer, the polylactic acid / retinoic acid electrospinning solution is sequentially electrospun onto the layer to obtain a sponge-nanofiber-nanofiber composite dressing.

2. A method for preparing a composite dressing for inhibiting scarring as claimed in claim 1, characterized in that: After the silk fibroin electrospinning was completed, the dressing surface was treated with ethanol vapor and then dried.

3. The method for preparing a composite dressing for inhibiting scarring according to claim 1, characterized in that: The preparation method of the CMC sponge layer is as follows: cotton fiber is treated with alkali solution and then reacted with a carboxymethylating agent, then the solution is dissolved with deionized water to obtain a CMC solution, the CMC solution is poured into a mold, and freeze-dried to obtain a highly absorbent CMC sponge.

4. A method for preparing a composite dressing for inhibiting scarring as claimed in claim 1, characterized in that: The silk fibroin electrospinning solution is obtained by dissolving silk fibroin in a hexafluoroisopropanol solution.

5. A method for preparing a composite dressing for inhibiting scarring as claimed in claim 4, characterized in that: The mass fraction of silk fibroin in the silk fibroin electrostatic spinning solution is 3-10%.

6. A method for preparing a composite dressing for inhibiting scarring as claimed in claim 1, characterized in that: The polylactic acid / retinoic acid electrospinning solution is obtained by adding polylactic acid and retinoic acid into a hexafluoroisopropanol solution in a certain proportion.

7. A method for preparing a composite dressing for inhibiting scarring as claimed in claim 6, characterized in that: The mass fraction of the polylactic acid is 5-15%, and the mass fraction of the retinoic acid is 0.01-0.5%.

8. The method for preparing a composite dressing for inhibiting scarring according to claim 1, characterized in that: The electrospinning process parameters of the silk fibroin electrospinning solution are: using a No. 20 spinning needle, a spinning voltage of 15 to 30 kV, a distance of 10 to 25 cm between the spinning needle and the receiver, a solution flow rate of 0.5 to 5 mL / h, and a spinning time of 10 to 60 min.

9. The method for preparing a composite dressing for inhibiting scarring according to claim 1, characterized in that: The electrospinning process parameters of the polylactic acid / retinoic acid electrospinning solution are: using a No. 20 spinning needle, a spinning voltage of 15 to 30 kV, a distance of 10 to 25 cm between the spinning needle and the receiver, a solution flow rate of 0.5 to 5 mL / h, and a spinning time of 10 to 60 min; adjusting the rotation speed of the collector to obtain nanofiber structures with different orientations; when the collector speed is set to 20 rpm to 100 rpm, randomly arranged nanofibers are obtained; and when the collector speed is set to 2000 rpm to 3000 rpm, orderedly arranged nanofibers are obtained.

10. The composite dressing obtained by the method according to any one of claims 1 to 9, characterized in that: The dressing comprises a highly water-absorbent CMC sponge outer layer, a hydrophilic silk fibroin nanofiber membrane, and an ordered polylactic acid / retinoic acid nanofiber inner layer.

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