Superfine-diameter PCL / COL nanofiber membrane material as well as preparation method and application thereof

Abstract

The invention provides a superfine-diameter PCL / COL nanofiber membrane as well as a preparation method and application thereof, and relates to the technical field of skin wound repair materials. The PCL / COL nanofiber membrane material is prepared from PCL and I-type collagen as raw materials through electrostatic spinning and vacuum drying. According to the preparation method disclosed by the invention, an electrostatic spinning technology is combined with strict regulation and control on parameters, so that the stable and superfine 40-nanoscale PCL / COL nanofiber membrane material is prepared. The diameter of the superfine PCL / COL nanofiber membrane material reaches the bionic scale of the minimum natural collagen of an extracellular matrix, and meanwhile, the superfine PCL / COL nanofiber membrane material shows more excellent hydrophilicity and lower surface roughness, has good biological safety and mechanical stability, not only realizes high matching with a natural ECM hierarchical structure, but also has good application prospects. By enhancing the cell-material interface interaction, the adhesion, proliferation and migration capacities of fibroblasts are remarkably improved, the wound healing period can be effectively shortened, an excellent repair promoting effect is shown, and a solid foundation is laid for clinical transformation of materials.

Description

Technical Field

[0001] The present invention relates to the technical field of skin wound repair materials, and particularly to an ultrafine diameter PCL / COL nanofiber membrane material, a preparation method thereof, and an application thereof. Background Art

[0002] Skin trauma refers to the situation where the integrity of the skin is damaged due to various injury-causing factors. According to the injury-causing factors, skin trauma can be divided into mechanical trauma (such as abrasions, contusions, lacerations, stab wounds, and avulsion injuries), physical trauma (such as burns, electric shock injuries, and radiation injuries), and chemical trauma (such as acid-base burns). According to the depth of the trauma, skin trauma can be divided into epidermal injury, dermal injury, and full-thickness skin injury. After trauma, the skin will undergo pathophysiological processes of the inflammatory reaction phase, tissue repair phase, and scar formation phase. The inflammatory reaction phase is mainly manifested by vasodilation, cell infiltration, and exudate formation; the tissue repair phase is mainly characterized by cell proliferation, matrix synthesis, and granulation tissue formation; and the scar formation phase is manifested by scar maturation and functional recovery.

[0003] Skin grafting is one of the effective methods for repairing skin trauma, but this method faces bottlenecks such as donor shortage, immune rejection, and insufficient functional reconstruction. Therefore, the development of new biomimetic repair materials has become the key to meeting the clinical needs for burn and scald repair.

[0004] The three-dimensional nanofiber membrane material prepared by electrospinning technology exhibits significant advantages in skin tissue engineering due to its unique biomimetic structural characteristics. This technology can construct a porous network with a high specific surface area, submicron fiber diameter, and high porosity, and its hierarchical fiber arrangement is highly similar to the natural extracellular matrix (ECM). It provides an ideal growth microenvironment for cell growth, can regulate cell adhesion, migration, and differentiation behaviors, and provides an innovative path for the development of new biomaterials in the fields of tissue engineering and regenerative medicine.

[0005] CN113786516A discloses a PCL / Col / MC gradient three-layer artificial perichondrium and its preparation method and application, belonging to the field of orthopedic implant materials, including the preparation of electrospinning solution and the preparation of perichondrium materials. This method prepares a three-layer composite fiber scaffold with gradually changing layers of PCL, Col, and MC components by electrospinning. Three consecutive electrospinnings are carried out using PCL, PCL / Col, and PCL / Col / MC solutions respectively, and when alternating the two solutions, 10% of the solution volume of both is electrospun simultaneously. It has a tensile strength similar to that of natural perichondrium, and each layer has its own function. The outer layer of the prepared artificial perichondrium is a pure PCL fiber layer, which has good soft tissue shielding function. The middle layer is PCL / collagen fiber, which provides mechanical support for the whole artificial perichondrium. The inner layer is a PCL / Col / MC composite fiber, which has good osteoinductive activity and osteoconductive ability. However, when this material is used for bone tissue regeneration, its thickness, breathability, and nutrient diffusion are limited, and its brittleness and high rigidity do not match the soft skin tissue seriously.

[0006] In addition, studies have shown that the diameter of nanofibers can significantly affect cell behavior, including adhesion, proliferation, and differentiation. For example, a study by Wu P et al. focused on the effect of nanofiber diameter on olfactory ensheathing cells (OECs). Using ramie silk fibroin nanofibers with diameters of 410 nm and 1200 nm respectively as membranes, the results showed that the fibers with smaller diameters were more conducive to cell adhesion, growth, and migration. This indicates that finer fibers may better mimic the extracellular matrix and provide a more favorable environment for cell proliferation and differentiation. In addition, a study on electrospun cellulose protein fibers by Hodgkinson T et al. found that fibers with smaller diameters (250 - 300 nm) could significantly support the proliferation of skin fibroblasts more than fibers with larger diameters (~1 μm). The smaller fibers promoted the formation of stellate cell morphology, increased the interaction between a large number of cells and fibers, and enhanced cell activities crucial for wound healing. This study emphasizes the importance of fiber diameter in designing membranes that effectively support cell growth and tissue regeneration.

[0007] Existing studies mostly use fibers with diameters of 100 - 500 nm to construct membranes. Although they can simulate some hierarchical structural characteristics of the natural extracellular matrix (ECM), there is a significant size deviation between their fiber diameters and the core components of the natural ECM (50 - 80 nm collagen fibers). In addition, existing fiber membranes have biological problems such as insufficient fibroblast adhesion efficiency and proliferation activity, which restrict their application efficiency in the field of tissue repair. Polycaprolactone (PCL) is a commonly used material for producing nanofibers, but it is still a challenge to prepare ultra-fine PCL nanofibers with diameters less than 100 nm.

[0008] Therefore, developing a skin wound repair material with a diameter less than 100 nm that can effectively optimize the cell microenvironment, shorten the wound healing cycle, and have excellent repair-promoting effects can overcome the problems existing in the prior art. Summary of the Invention

[0009] In view of this, the present invention provides an ultra-fine diameter polycaprolactone (PCL) / type I collagen (COL) nanofiber membrane material, its preparation method and application. The present invention combines electrospinning technology with strict control of parameters to endow the nanofiber membrane material with both biocompatibility and mechanical stability, and uses this to optimize the cell microenvironment. It not only achieves a high degree of matching with the hierarchical structure of natural ECM, but also significantly improves the spreading, proliferation and migration ability of fibroblasts by enhancing the cell-material interface interaction, laying a solid foundation for the clinical transformation of the material.

[0010] The first aspect of the present invention is to provide an ultra-fine diameter PCL / COL nanofiber membrane material, which is obtained by electrospinning and vacuum drying using PCL and type I collagen as raw materials; The diameter of the PCL / COL nanofiber membrane material is (41.4±6.5) nm.

[0011] The second aspect of the present invention is to provide a preparation method of an ultra-fine diameter PCL / COL nanofiber membrane material, including the following steps: Dissolve PCL and type I glue in HFIP to obtain a spinning solution, and the spinning solution is electrospun and vacuum dried to obtain a PCL / COL nanofiber membrane material.

[0012] Preferably, in the spinning solution, the total content of PCL and COL is 2 wt.%, and the mass ratio of PCL to COL is 3:1.

[0013] Preferably, the electrospinning parameters are set as follows: the flow rate is 3-9 μL / min, the voltage is 15-19 kV, the receiving distance is 10-20 cm, the needle size is 20-24 G, the air humidity < 30%, and the environmental temperature is 30°C - 35°C. More preferably, the electrospinning parameters are set as follows: the flow rate is 3 μL / min, the voltage is 19 kV, the receiving distance is 10 cm, and the needle size is 24 G.

[0014] Preferably, the collector is an aluminum foil collector.

[0015] Preferably, the vacuum drying temperature is 50°C, and the drying time is 24-48 h.

[0016] The third aspect of the present invention is to provide an application of an ultra-fine diameter PCL / COL nanofiber membrane material as a skin wound repair material.

[0017] Compared with the prior art, the beneficial technical effects of the present invention are as follows: The present invention adopts the PCL / COL blend electrospinning process. By systematically optimizing the spinning parameters, a stable ultrafine diameter PCL / COL nanofiber membrane material is prepared. The ultrafine diameter PCL / COL nanofiber membrane material of the present invention reaches the biomimetic scale of the smallest natural collagen in the extracellular matrix, and at the same time exhibits better hydrophilicity and lower surface roughness, has good biosecurity, can effectively promote cell spreading, proliferation and migration, shorten the wound healing cycle, and shows excellent repair-promoting effects. Brief Description of the Drawings

[0018] The present invention will be further described below in conjunction with the drawings.

[0019] Figure 1 is the SEM image of the PCL / COL nanofiber membrane material; Figure 2 is the diameter distribution of the PCL / COL nanofiber membrane material; Figure 3 is the Fourier transform infrared spectrum of the PCL / COL nanofiber membrane material; Figure 4 is the water contact angle measurement result; Figure 5 is the AFM image of the PCL / COL nanofiber membrane material; Figure 6 is the roughness detection result of the PCL / COL nanofiber membrane material; Figure 7 is the biocompatibility detection result; Figure 8 is the cell morphology and spreading detection result; Figure 9 is the proliferation ability detection result of cells on the PCL / COL nanofiber scaffold material; Figure 10 is the migration ability detection result of cells on the PCL / COL nanofiber membrane material; Figure 11 is the wound healing situation; Figure 12 is the wound healing rate statistics; Figure 13 is the nanofiber when the mass-volume percentage of PCL / COL is reduced to 1.5%. Detailed Embodiments

[0020] Next, in combination with the embodiments of the present invention, the technical solutions of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0021] The first aspect of the present invention is to provide an ultrafine diameter PCL / COL nanofiber membrane material, which is obtained by electrospinning and vacuum drying using PCL and type I collagen as raw materials. The diameter of the PCL / COL nanofiber membrane material is (41.4 ± 6.5) nm.

[0022] The second aspect of the present invention is to provide a preparation method of an ultrafine diameter PCL / COL nanofiber membrane material, including the following steps: S1. Add PCL and COL into HFIP, stir until completely dissolved, and then defoam to obtain a spinning solution. S2. Use the spinning solution in step S1 as the raw material for electrospinning, receive the nanofiber membrane material with a collector plate, and then vacuum dry to obtain the PCL / COL nanofiber membrane material.

[0023] In the spinning solution of the present invention, the mass ratio of PCL to COL is 3:1, and the content of PCL and COL in the spinning solution is 2%. The electrospinning parameters are set as follows: the flow rate is 3 - 9 μL / min, preferably 3 μL / min; the voltage is 15 - 19 kV, preferably 19 kV; the receiving distance is 10 - 20 cm, preferably 10 cm; the needle gauge is 20 - 24 G, preferably 24 G; the air humidity < 30%, the environmental temperature is 30°C - 35°C, and the collector is an aluminum foil collector; the drying temperature is 50°C, and the drying time is more than 24 h. However, the present invention does not strictly limit the drying time, and the specific drying time can be appropriately adjusted according to the water content of the sample as long as the material is sufficiently dried.

[0024] As a semi-crystalline polyester material, PCL has good mechanical properties and biocompatibility, but its hydrophobic surface limits cell adhesion. Collagen (COL), as the main component of ECM, has excellent biocompatibility. Type I collagen (CollagenI, COL) is the main collagen in most tissues of higher animals.

[0025] The present invention realizes the synergistic modification of the two materials through electrospinning technology and parameter limitation. Among them, the introduction of PCL improves the mechanical strength of the membrane material, and the addition of COL improves the hydrophilicity of the material, making the composite membrane material have both biocompatibility and mechanical stability.

[0026] The present invention strictly defines the electrospinning process. The electrospinning process of the present invention is strictly applicable to the technical solution of the present invention, which can achieve the best combination of PCL and COL, and precisely control the fiber diameter to the biomimetic scale (40 nanometers) of the smallest natural collagen in the extracellular matrix, effectively optimizing the cell microenvironment. The ultrafine fiber membrane of the present invention not only achieves a high degree of matching with the hierarchical structure of natural ECM, but also significantly improves the adhesion, proliferation and migration ability of fibroblasts by enhancing the cell-material interface interaction, providing a new solution for biomedical fields such as skin tissue engineering, bone repair and nerve regeneration.

[0027] The third aspect of the present invention is to provide an application of an ultrafine diameter PCL / COL nanofiber membrane material as a skin wound repair material.

[0028] The present invention does not strictly limit the usage method of the ultrafine diameter PCL / COL nanofiber membrane material, and the conventional usage methods of skin wound repair materials in the art are applicable to this material.

[0029] For example, after debridement, the wound surface is rinsed with normal saline to remove necrotic tissue and stop bleeding, ensuring that the wound microenvironment is moist. Before use, it is sterilized by ethylene oxide or ultraviolet light. According to the shape of the wound surface, the PCL / COL nanofiber membrane material is cut into an appropriate size (usually slightly larger than the wound edge by 1-2 mm), and the fiber membrane (single layer or multiple layers stacked) is directly applied to the wound surface, and the outer layer is supplemented with a medical silicone film or breathable gauze for pressure dressing. Then it can be replaced regularly according to the wound condition.

[0030] To further illustrate the present invention, the following examples are provided for detailed description. The raw materials used in the following examples of the present invention are all commercially available.

[0031] Unless otherwise specified, all experiments are repeated 3 times. ANOVA and Duncan multiple comparison analysis are performed using SPSS 21.0, and the results are expressed as mean ± standard deviation. A significant difference is indicated when P < 0.05.

[0032] Example 1 Preparation method of PCL / COL nanofiber membrane material, the steps are as follows: S1. Weigh PCL and COL according to the mass ratio of PCL:COL of 3:1, add PCL and COL to the solvent HFIP, and magnetically stir for 24 h to completely dissolve them, preparing mixed raw materials with a mass-volume percentage of 2% (Sample A), 8% (Sample B), and 12% (Sample C). Let it stand for 24 h to remove the solution bubbles, and set aside for later use; S2. Place the experimental solution in a 10 mL disposable syringe, install the needle according to the sample. Use 24G for Sample A, 22G for Sample B, and 20G for Sample C; Parameter setting: Flow rate: 3 μL / min for sample A, 6 μL / min for sample B, 9 μL / min for sample C; Voltage: 19 kV for sample A, 17 kV for sample B, 15 kV for sample C; Receiving distance between the needle tip and the aluminum foil collector plate: 10 cm for sample A, 15 cm for sample B, 20 cm for sample C; Air humidity: < 30%, ambient temperature: 30°C - 35°C; Load the degassed solution into the syringe and connect the device, then turn on the high-voltage power supply. After a stable Taylor cone and fine stream are formed at the needle tip, use aluminum foil to receive the nanofiber membrane material until a fiber membrane of the same thickness is formed. Vacuum dry the fiber membrane for more than 24 h to completely remove the residual hexafluoroisopropanol, and three different PCL / COL nanofiber membrane materials are obtained.

[0033]

[0034] Test Example 1 Surface Morphology Characterization After cleaning the surface of the PCL / COL nanofiber membrane material in Example 1, cut it into a square size of 1 cm × 1 cm and paste it on the detection platform. Cut and paste 3 pieces of the membrane material in the same group. After sputtering with gold, use a field emission scanning electron microscope (SEM) to observe and photograph the morphology of the nanofiber membrane material, and use ImageJ software to measure and statistically analyze the fiber diameter in the SEM images. The results are as Figure 1 shown.

[0035] As Figure 1 can be seen, the surface of the PCL / COL composite fiber is smooth and the diameter is uniform, presenting a typical three-dimensional connected network structure. Randomly select 20 fibers for measurement and statistics using ImageJ image analysis software.

[0036] The diameter of the electrospun nanofibers decreases as the mass-volume percentage (wt%) of PCL / COL decreases. When the mass-volume percentage (wt%) of PCL / COL decreases to 1.5%, continuous, bead-free, and uniformly-diameter nanofibers can no longer be obtained ( Figure 13 ).

[0037] After detection, the diameters of the PCL / COL nanofiber membrane materials of Sample A (2%), Sample B (8%), and Sample C (12%) were (41.4±6.5) nm, (255.8±35.7) nm, and (792.5±81.0) nm, respectively. Therefore, the nanofiber membrane material corresponding to Sample A was denoted as USNF (Ultra-Small Diameter Nanofibers), that is, the ultra-fine diameter PCL / COL nanofiber membrane material, the nanofiber membrane material corresponding to Sample B was denoted as MNF (Medium Diameter Nanofibers), and the nanofiber membrane material corresponding to Sample C was denoted as LNF (Large Diameter Nanofibers).

[0038] Test Example 2 Determination of Surface Chemical Groups After cleaning the surface of the PCL / COL nanofiber membrane material in Example 1, it was cut into a size of 1 cm×1 cm and placed into a Fourier transform infrared spectrometer (FTIR) for detection under the set mode and parameters. Each group of samples was measured 3 times repeatedly, and the results are as Figure 3 shown.

[0039] It can be seen from Figure 3 that the absorption peaks in the infrared spectra of the three PCL / COL nanofiber membrane materials all include the characteristic absorption peaks of PCL (1726 cm -1 , 1241 cm -1 ) and COL (3326 cm -1 , 1659 cm -1 ), which confirmed the existence of PCL and COL. However, no absorption peak appeared or the peak position shifted, indicating that no chemical reaction occurred between PCL and COL, and only physical cross-linking occurred.

[0040] Test Example 3 Hydrophilicity and Hydrophobicity Test The water contact angle (WCA) is a core parameter characterizing the surface wettability of materials, and its measurement system has important value in evaluating the hydrophilicity and hydrophobicity of electrospun membrane materials. Drop the liquid on the surface of the PCL / COL composite nanofiber membrane material to be tested in Example 1, obtain the shape image of the liquid drop through the microscopic lens and camera of the contact angle analyzer, and use digital image algorithms to process and calculate the contact angle size of the liquid drop in the captured image, and indirectly reflect the hydrophilicity and hydrophobicity of the measured membrane material by the size of the water contact angle. Each group of samples was measured 4 times repeatedly, and the results are as Figure 4 shown.

[0041] It can be seen from Figure 4It can be seen that the average values of the measured results of the water contact angle of the samples are 71.3°, 104.6°, and 114.5° respectively. The hydrophilicity of the fiber membrane increases with the decrease of the fiber diameter. Among them, the 40nm ultra-fine PCL / COL nanofiber membrane (USNF) has the smallest surface contact angle and better hydrophilicity.

[0042] Test Example 4 Determination of the original surface roughness After cleaning the surface of the PCL / COL nanofiber membrane material in Example 1, it was cut into a square size of 2 cm × 2 cm. The detection mode was set to dynamic detection and contact mode. Three 1 cm × 1 cm areas were selected on the surface of the sample to be tested, and the detection speed was 0.5 In / s. The results are as Figures 5 - 6 shown.

[0043] Experiments found that the roughness of the fiber membrane decreases with the decrease of the fiber diameter. Compared with the membrane materials of the control group diameter specifications, the 40 nm ultra-fine PCL / COL nanofiber membrane material (USNF) shows lower surface roughness values. The arithmetic mean roughness (Rq) of the three materials USNF, MNF, and LNF are 0.098, 0.166, and 0.241 respectively, and the root mean square roughness (Ra) are 0.078, 0.131, and 0.195 respectively.

[0044] Test Example 5 Biocompatibility evaluation Fresh whole blood of healthy experimental animals (New Zealand rabbits) was aseptically collected. After anticoagulation treatment, red blood cells were separated by centrifugation at 3000 rpm for 15 min. The red blood cells were washed 3 times with pre-cooled 0.9% sodium chloride solution (PBS) to remove plasma residues. The red blood cells were resuspended in PBS to prepare a 4% red blood cell suspension, which was mixed with the extraction solution of the nanomaterial to be tested (Samples A / B / C) at a volume ratio of 1:2, and incubated at 37°C for 1 h (oscillation rate 60 rpm). A negative control (PBS) and a positive control (0.1% TritonX-100) were set up synchronously. After incubation, centrifugation was carried out at 2000 rpm for 10 min, and 100 μL of the supernatant was transferred to a 96-well plate. The absorbance (OD value) was measured at 541 nm using a Tecan Infinite 200PRO microplate reader, and 3 replicates were set for each group.

[0045] The preparation method of the extraction solution is as follows: The PCL / COL nanofiber membrane (3 cm 2 / mL) was immersed in PBS and extracted at 37°C for 24 h (the volume ratio of the material to the solvent is 1:10). After centrifugation and filtration, the extraction solution was obtained.

[0046] Hemolysis rate = ([A]test - [A]negative) / ([A]positive - [A]negative) × 100%.

[0047] The detection results are asFigure 7 as shown

[0048] As can be seen from Figure 7 it, the nanofiber membrane materials of A / B / C samples do not cause red blood cell damage. Compared with the negative control (PBS), the hemolysis rates of the three material samples are all relatively low (<5.0%), indicating that the materials have good blood compatibility.

[0049] Test Example 6 Cell Morphology, Spreading, Migration, and Proliferation on Nanofiber Membrane Materials Normal human dermal fibroblasts (NHDFs) were seeded onto the surfaces of the membrane materials prepared in Examples 1 to 3 after pretreatment at a standard density (about 1×10 5 cells / cm 2 ), and then cultured routinely in an incubator at 37°C and 5% CO2 for 72 h. They were gently rinsed 3 times with PBS buffer, fixed with 2.5% glutaraldehyde at room temperature for 60 min, and then washed 3 times with PBS (20 min each time) to remove the residual fixative. They were dehydrated successively through gradients of 70%, 80%, 90%, 95%, and 100% ethanol, air-dried, sputter-coated with gold, and finally observed for morphological structure by scanning electron microscopy. The results are as Figure 8 shown

[0050] The material pretreatment method is as follows: After the materials are fully dried, they are irradiated at 60°C (2×10 4 Gy) for 2 h, soaked in 75% ethanol for 30 min, rinsed 3 times with PBS, and incubated overnight with the culture medium in an incubator at 37°C.

[0051] As can be seen from Figure 8 it, the fibroblasts seeded onto the surface of the 40 nm - grade ultra - fine PCL / COL nanofiber membrane (USNF) prepared in Example 1 showed the largest spreading area, and the cell morphological characteristics were significantly better than those of other experimental groups.

[0052] NHDFs cells were seeded onto three nanofiber membrane materials with different diameters. After culturing for 1, 3, 5, and 7 d, the cell proliferation ability was detected by CCK - 8. The results are as Figure 9 shown. As can be seen from Figure 9 it, as the fiber diameter increases, the cell proliferation ability decreases.

[0053] Inserts with a width of 0.9 mm were placed on the surfaces of the three nanofiber membrane materials. NHDFs cells were seeded on both sides of the inserts. After 12 h, the inserts were removed to form a cell wound with a width of 0.9 cm. After staining and photographing at 0, 12, 24, 48 h after culturing, the cell migration ability was detected. The results are as Figure 10as shown

[0054] From Figure 10 it can be seen that the cell migration ability of cells growing on USNF is significantly higher than that of cells growing on the other two fibers, and the difference is statistically significant. The cell wound area was statistically analyzed to clarify the effect of the ultrafine nanofiber membrane material on cell migration ability. NHDFs cells were seeded on the surface of the pretreated membrane material at a density of 5×10 5 cells / cm 2 and cultured routinely in an incubator at 37°C and 5% CO2 for more than 24 h. The experimental results showed that the cell migration ability of the 40 nm ultrafine PCL / COL nanofiber membrane group (USNF) prepared in Example 1 was faster than that of the control group.

[0055] Live cell real-time tracking analysis showed that the linearity of the cell movement trajectories, the straight-line ratio of the cell movement trajectories and the movement rate of the 41 nm fiber membrane group were all significantly better than those of the other experimental groups. [[ID=1�]]

[0056] Test Example 7 In Vivo Wound Healing Evaluation An SD rat full-thickness skin defect model was established as follows: SPF-grade 8-week-old male SD rats were selected. After anesthesia, a full-thickness skin wound reaching the deep fascia with a size of 1.0 cm×1.0 cm was made in the middle of the back of the rats. The rats were randomly divided into four groups: the sterile dressing covering group (Blank Control), the ultrafine nanofiber membrane covering group (USNF), the medium nanofiber membrane covering group (MNF), and the large-diameter nanofiber membrane covering group (LNF). Different types of fiber membranes were covered on the wounds, and the coating amount and coating thickness of the fiber membranes in each group were ensured to be the same. Then, sterile dressings and oil gauzes were covered in turn. After 0, 3, 7, and 14 days of covering the wounds with the nanofiber membranes, photos were taken with a digital camera to record the wound healing situation, and the immediate wound area of each group was analyzed using the image analysis software Image-Pro Plus, and the wound healing rate was calculated.

[0057] Healing rate = (immediate wound area / original wound area) × 100%.

[0058] The results are as Figure 11 、 Figure 12 shown.

[0059] From Figure 11 、 Figure 12 it can be seen that the wound healing speed of the 40 nm PCL / COL ultrafine nanofiber membrane group (USNF) is significantly higher than that of the other three groups (P<0.05), and the 40 nm PCL / COL ultrafine nanofiber membrane group (USNF) shows excellent repair-promoting efficacy.

[0060] Comparative Example 1 Same as Example 1, with the difference that COL is replaced by an equal amount of keratin.

[0061] The results show that the fiber membrane material meeting the standards cannot be obtained by this solution.

[0062] Due to strong intermolecular hydrogen bonding and lack of plasticity in the pure keratin solution, bead nodes are formed in the electrospinning process of the present invention. Eventually, the obtained fiber diameter is uneven and brittle, and the yield rate is extremely low, making it unusable.

[0063] Taking a small amount of qualified samples for performance testing, it is found that the fiber cell adhesion rate decreases, the hydrophobicity is relatively strong (contact angle > 90°), and the stability of the spinning solution is poor (shelf life < 3 days, while the spinning solution in Example 1 can be stably stored for more than 7 days).

[0064] Comparative Example 2 According to the content recorded in CN106049026A, a PCL / COL composite fiber membrane is prepared by the dipping method, and the specific method is as follows: Under the constant temperature condition of 4°C, type I collagen is dissolved in PBS buffer solution with a pH of 7.2 to prepare a 5 mg / mL type I collagen solution. Then, the ultrasonically treated PCL nanofiber membrane is immersed in the type I collagen solution for 2 h, and finally dried on a sterile clean workbench.

[0065] The raw materials for preparing the PCL nanofiber membrane material are the same as those in Example 1A, with the difference being the dipping method.

[0066] The results show that compared with the USNF in Example 1, the COL of the fiber material obtained in this comparative example is enriched on the surface of the PCL membrane, and there is almost no COL penetration inside, forming a "sandwich layer" structure, which is only suitable for surface functionalization requirements (such as short-term antibacterial coating). While the nanofiber membrane prepared by the blending method in Example 1 has a complete three-dimensional network structure, can meet the requirements of complex tissue engineering (such as bone-cartilage gradient scaffolds, wound dressings), and the mechanical properties and degradation rate are controllable (7 - 30 days). In addition, the composite fiber membrane obtained in this comparative example is only combined by physical adsorption, with low interfacial bonding strength, delamination occurs in a humid environment, and a strong interfacial bond cannot be formed, resulting in a significant reduction in tensile strength and water resistance.

[0067] The above-mentioned embodiments only represent several implementation manners of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several deformations and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. An ultrafine diameter PCL / COL nanofiber membrane material, characterized in that, The nanofiber membrane material is obtained by electrospinning and vacuum drying using PCL and type I collagen as raw materials; The diameter of the PCL / COL nanofiber membrane material is (41.4±6.5) nm.

2. A method for preparing the ultrafine diameter PCL / COL nanofiber membrane material according to claim 1, characterized in that, It includes the following steps: Dissolve PCL and type I collagen in HFIP to obtain a spinning solution. The spinning solution is electrospun and vacuum dried to obtain the PCL / COL nanofiber membrane material.

3. The preparation method according to claim 2, wherein In the spinning solution, the total content of PCL and COL is 2 wt.%.

4. The preparation method according to claim 2, characterized in that, In the spinning solution, the mass ratio of PCL to COL is 3:

1.

5. The preparation method according to claim 2, characterized in that, The electrospinning parameters are set as follows: the flow rate is 3-9 μL / min, the voltage is 15-19 kV, the receiving distance is 10-20 cm, the needle gauge is 20-24G, the air humidity < 30%, and the ambient temperature is 30°C - 35°C.

6. The preparation method according to claim 5, wherein The electrospinning parameters are set as follows: the flow rate is 3 μL / min, the voltage is 19 kV, the receiving distance is 10 cm, and the needle gauge is 24G. The electrospinning parameters are set as follows: the flow rate is 3 μL / min, the voltage is 19 kV, the receiving distance is 10 cm, and the needle gauge is 24G.

7. The preparation method according to claim 2, characterized in that, The collector in the electrospinning process is an aluminum foil collector.

8. The preparation method according to claim 2, characterized in that, The vacuum drying temperature is 50°C, and the drying time is 24-48 h.

9. Use of an ultra-fine diameter PCL / COL nanofiber membrane material as a skin wound repair material, characterized in that, The ultrafine diameter PCL / COL nanofiber membrane material is the ultrafine diameter PCL / COL nanofiber membrane material described in claim 1 or the ultrafine diameter PCL / COL nanofiber membrane material prepared by the method described in any one of claims 2-8.

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