PLCL / ADM composite nano short fibers and their preparation method and application

Abstract

The present invention provides a PLCL / ADM composite nanofiber, its preparation method, and application. The PLCL / ADM composite nanofiber is formed by combining PLCL and ADM to form nanofibers. The ratio of PLCL and ADM in the nanofibers is controlled to achieve a synergistic effect between the two. This ensures that when used in wound repair, the injectability of the hydrogel is not affected. Furthermore, the composite nanofiber gradually releases various effective substances to promote collagen production, accelerate wound healing, and improve wound repair effectiveness.

Description

Technical Field

[0001] The present invention belongs to the technical field of wound repair, and in particular relates to a PLCL / ADM composite nano short fiber and a preparation method and application thereof. Background Art

[0002] A wound is damage to normal skin (tissue) caused by external factors such as surgery, force, heat, electric current, chemicals, and low temperatures, as well as internal factors such as local blood supply disruption. It is often accompanied by a loss of skin integrity and a certain amount of normal tissue, while also impairing the skin's normal functions. It is also called a wound or trauma. Wound repair is also known as wound repair.

[0003] Traditionally, wounds are repaired using a dressing and gauze wrapping method, but the repair effect needs to be improved. Furthermore, traditional dressings can easily tear the wound during dressing changes. Summary of the Invention

[0004] In order to solve or at least partially solve the above-mentioned defects, the present invention provides a PLCL / ADM composite nano-short fiber and a preparation method and application thereof.

[0005] The first aspect of the present invention is to provide a PLCL / ADM composite nano short fiber, wherein the mass ratio of PLCL to ADM is (3-7): (3-7).

[0006] Furthermore, the diameter of the PLCL / ADM composite nanofiber is (680±270) nm;

[0007] The aspect ratio of the PLCL / ADM composite nano short fibers is (950-410): (67-5).

[0008] A second aspect of the present invention provides a method for preparing the above-mentioned PLCL / ADM composite nanofibers, comprising the following steps:

[0009] Preparation of PLCL / ADM composite nanofibers;

[0010] homogenizing the PLCL / ADM composite nanofibers to obtain a nanofiber dispersion;

[0011] The nano-short fiber dispersion is freeze-dried to prepare PLCL / ADM composite nano-short fibers.

[0012] Furthermore, the homogenization speed is 5000rpm-10000rpm, and the time is 30min-60min;

[0013] The freeze-drying temperature is -90°C to -70°C. Preferably, the nano-short fiber dispersion is first frozen at -30°C to -10°C for 0.5h to 2h, then super-frozen at -90°C to 70°C, and then freeze-dried for 45h to 50h.

[0014] Furthermore, the preparation of PLCL / ADM composite nanofibers comprises the following steps:

[0015] Dissolve PLCL and ADM in a solvent to prepare a PLCL / ADM mixed spinning solution:

[0016] Electrospinning the PLCL / ADM mixed spinning solution to obtain PLCL / ADM composite nanofibers;

[0017] Preferably, dissolving PLCL and ADM in a solvent to form a PLCL / ADM mixed spinning solution comprises: dissolving PLCL and ADM in a mass ratio of (3-7): (3-7) in hexafluoroisopropanol at a mass volume ratio of 5-15% to form a PLCL / ADM mixed spinning solution; and / or, the voltage of the electrospinning is 5kV-15kV, and the flow rate is 1mL / h-3mL / h.

[0018] The third aspect of the present invention provides a use of the above-mentioned PLCL / ADM composite nano-short fibers or the PLCL / ADM composite nano-short fibers prepared by the above-mentioned preparation method in a wound repair preparation.

[0019] The fourth aspect of the present invention provides a PLCL / ADM composite nano-short fiber containing the above-mentioned PLCL / ADM composite nano-short fiber or the PLCL / ADM composite nano-short fiber prepared by the above-mentioned preparation method.

[0020] A fifth aspect of the present invention provides a wound repair preparation comprising the following ingredients:

[0021] (1) the above-mentioned PLCL / ADM composite nanofibers or the PLCL / ADM composite nanofibers prepared by the above-mentioned preparation method;

[0022] (2) GelMA solution;

[0023] Furthermore, the mass volume ratio of the PLCL / ADM composite nanofibers to the GelMA solution is 1-6% (w / v); preferably, the degree of substitution of the GelMA is 30%-90%, and the molecular weight is 100kDa-200kDa;

[0024] Furthermore, ADSCs are included, and the volume ratio of the ADSCs to the GelMA solution is 0.5*10 4 —1*104 cells.

[0025] A sixth aspect of the present invention provides a process for preparing the wound repair preparation, comprising the following steps:

[0026] Prepare a GelMA solution with a mass concentration of 5-30%;

[0027] The PLCL / ADM composite nanofibers are added to the GelMA solution and mixed to obtain a wound repair preparation.

[0028] Furthermore, the method further comprises resuspending the ADSCs cell pellet by the mixed solution obtained by mixing, and blowing evenly to prepare the wound repair preparation, wherein the mass volume ratio of the PLCL / ADM composite nanofibers and the GelMA solution is 1-6% (w / v), and the number volume ratio of the ADSCs to the GelMA solution is 0.5*10 per 1 mL of the GelMA solution. 4 —1*10 4 cells.

[0029] Furthermore, the GelMA solution having a mass concentration of 5-30% comprises:

[0030] Prepare a 0.2% (w / v)-0.3% (w / v) initiator standard solution;

[0031] The GelMA was added to the initiator standard solution, and heated in a water bath at 60-80° C. in the dark for 25-35 min to dissolve the solution, with shaking during the process to allow the GelMA to fully infiltrate the solution, to prepare a GelMA solution with a mass concentration of 5-30%.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The PLCL / ADM composite nanofibers provided in a specific embodiment of the present invention are nanofibers formed by compounding PLCL and ADM, and controlling the ratio of PLCL and ADM in the nanofibers to exert a synergistic effect between the two, ensuring that when used in wound repair, the injectability of the hydrogel is not affected, and the nanofibers can gradually release various effective substances to promote collagen production, accelerate wound healing, and improve the wound repair effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0035] Figure 1 The electron microscope image and Fourier infrared spectrum image of the PLCL / ADM composite nanofiber scaffold in the embodiment of the present invention are shown, wherein: (A) electron microscope image of the PLCL / ADM composite nanofiber scaffold; (B) Fourier infrared spectrum image of the PLCL / ADM composite nanofiber scaffold;

[0036] Figure 2 This is a bar graph obtained from a cell proliferation experiment in which fibroblasts were seeded on a PLCL / ADM composite nanofiber scaffold and a blank glass slide in an embodiment of the present invention;

[0037] Figure 3 : These are comparison diagrams of the bioactive hydrogel dressing before and after light curing according to an embodiment of the present invention, wherein (A) is a photo of the dressing before light curing, and (B) is a photo of the gel after light curing and cross-linking.

[0038] Figure 4 This is a bar graph obtained from a cell proliferation experiment of the wound repair agent prepared in Example 4 of the present invention and GelMA-loaded ADSCs;

[0039] Figure 5 : These are diagrams illustrating a full-thickness skin defect model in rats according to an embodiment of the present invention, including: (A) a diagram illustrating the wound healing process in a full-thickness skin defect model in rats; and (B) a bar graph illustrating the calculated wound healing rate.

[0040] Figure 6 : HE staining image of wound healing tissue on day 7 and a histogram obtained by counting the epidermal thickness of the corresponding healed wound in an embodiment of the present invention, wherein: (A) HE staining image of wound healing tissue on day 7; (B) histogram obtained by counting the epidermal thickness of the healed wound;

[0041] Figure 7 The following are Masson staining images of wound healing tissue on day 7 and histograms of the collagen content of the corresponding healed wounds according to the embodiment of the present invention, wherein: (A) HE staining image of wound healing tissue on day 7; (B) histogram of the collagen content of the healed wounds;

[0042] Figure 8 The figure below shows the mechanical properties of the hydrogels in the embodiment of the present invention. The left figure shows the shear properties before photocrosslinking, and the right figure shows the stress-strain changes after crosslinking.

[0043] Figure 9 This is a bar graph obtained from a cell proliferation experiment in which fibroblasts were seeded on a PLCL / ADM composite nanofiber scaffold, a blank glass slide, and a PLCL nanofiber membrane in an embodiment of the present invention;

[0044] Figure 10 It is a degradation curve diagram of the PLCL / ADM composite nanofiber scaffold and the PCL / ADM composite nanofiber scaffold in the embodiment of the present invention. DETAILED DESCRIPTION

[0045] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0046] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0047] PLCL (poly-L-lactic acid-caprolactone) was purchased from Shenzhen Maiqi Biomaterial Co., Ltd.; ADM (acellular dermal matrix) was purchased from Qingdao Institute of Marine Biomedicine, Ocean University of China; and hexafluoroisopropanol was purchased from Shanghai Darui Chemical Co., Ltd.

[0048] In order to prove that ADSCs (stem cells derived from adipose tissue) can be observed under a fluorescence microscope in pathological sections in animal experiments, it was proved that they survived and were effective. In the present invention, CM-Dil fluorescent labeling of ADSCs was used for staining, and the specific steps were as follows: 50 μg of CM-Dil labeling solution was added to 50 μL of dimethyl sulfoxide, and then D-PBS was added dropwise to prepare a working staining solution with a concentration of 1 μg / mL. Mouse ADSCs (HyCyte, P5) were grown adherently in a cell culture dish and washed 3 times with PBS. The working staining solution was added to the cell culture dish to cover the cells, incubated in a 37°C incubator in the dark for 5 minutes, and incubated in a 4°C refrigerator in the dark for 20 minutes. The working staining solution was removed and washed 3 times with PBS. The cells were digested with 0.25% trypsin, the cell suspension was centrifuged, the supernatant was removed, and a precipitate of mouse ADSCs labeled with CM-Dil labeling solution was obtained.

[0049] A specific embodiment of the present invention provides a PLCL / ADM composite nano-short fiber, wherein the mass ratio of PLCL to ADM is (3-7): (3-7), specifically, the mass ratio of PLCL to ADM can be 7:3, 5:5, or 3:7.

[0050] In certain specific embodiments, the diameter of the PLCL / ADM composite nanofibers is (680±270) nm, which represents the average diameter ± standard deviation, and may be, for example, 420 nm, 680 nm, 780 nm, 1000 nm, etc.; the aspect ratio of the PLCL / ADM composite nanofibers is (950-410):(67-5), where the aspect ratio refers to the ratio of the length to the diameter of the PLCL / ADM composite nanofibers, and may be, for example, 950:67, 950:5, 600:67, 410:5, etc. This configuration can further enhance the loading effect of the PLCL / ADM composite nanofibers in the GelMA solution without affecting their injectability, and can be effectively released in a short time. At the same time, this structure can also further enhance the mechanical properties of the GelMA solution.

[0051] In addition, a specific embodiment of the present invention provides a method for preparing the above-mentioned PLCL / ADM composite nanofibers, comprising the following steps:

[0052] Preparation of PLCL / ADM composite nanofibers;

[0053] homogenizing the PLCL / ADM composite nanofibers to obtain a nanofiber dispersion;

[0054] The nano-short fiber dispersion is freeze-dried to prepare PLCL / ADM composite nano-short fibers.

[0055] Compared with common loading forms such as nanospheres, the preparation method of the above-mentioned PLCL / ADM composite nanofibers is simple and efficient.

[0056] In some specific embodiments, the homogenization speed is 5000 rpm-10000 rpm, for example, 5000 rpm, 7000 rpm, 9000 rpm, 10000 rpm, and the time is 30 min-60 min, for example, 30 min, 40 min, 60 min;

[0057] The freeze-drying temperature is -90°C to -70°C. Preferably, the nano-short fiber dispersion is first pre-frozen at -30°C to -10°C for 0.5-2 hours, then super-frozen at -90°C to 70°C, and then freeze-dried for 45-50 hours. More preferably, the nano-short fiber dispersion is pre-frozen at -20°C for 0.5-2 hours, then super-frozen at -80°C, and then freeze-dried for 45-50 hours. The purpose of the pre-freezing + super-freezing method is to maintain the dispersion morphology of the short fibers in the homogenous liquid before freeze-drying, facilitating the dispersion of the short fibers after freeze-drying. The purpose of freeze-drying is to remove the liquid in the homogenous liquid, leaving only the short fibers.

[0058] Specifically, the preparation of PLCL / ADM composite nanofibers comprises the following steps:

[0059] Dissolve PLCL and ADM in a solvent to prepare a PLCL / ADM mixed spinning solution:

[0060] The PLCL / ADM mixed spinning solution is subjected to electrostatic spinning to prepare PLCL / ADM composite nanofibers.

[0061] In some specific embodiments, dissolving PLCL and ADM in a solvent to prepare a PLCL / ADM mixed spinning solution comprises: dissolving PLCL and ADM in a mass ratio of (3-7): (3-7) in hexafluoroisopropanol at a mass volume ratio of 5-15% to prepare a PLCL / ADM mixed spinning solution; and / or,

[0062] The voltage of the electrospinning is 5 kV-15 kV, for example, 5 kV, 10 kV, or 15 kV, and the flow rate is 1 mL / h-3 mL / h, for example, 1 mL / h, 2 mL / h, or 3 mL / h.

[0063] In some specific embodiments, the method for preparing the PLCL / ADM composite nanofibers comprises the following steps:

[0064] (1) Preparation of PLCL / ADM composite nanofiber scaffolds: PLCL / ADM mixed spinning solutions were prepared, wherein PLCL:ADM = 7:3, 5:5, and 3:7, and PLCL with a Mw of ≈ 300,000 (LLA:CL = 50:50) were dissolved in hexafluoroisopropanol (HFIP) at a mass volume ratio of 5-15%, and magnetically stirred overnight;

[0065] Using a common flat plate as a receiving device, the PLCL / ADM mixed spinning solution was sucked into a syringe, connected to a 21-gauge stainless steel needle and connected to a 5kV-15kV high voltage, and electrospinning was performed at a flow rate of 1mL / h-3mL / h to prepare PLCL / ADM composite nanofiber scaffolds with different ratios. The purpose of this step is to prepare PLCL / ADM composite nanofibers in the subsequent preparation;

[0066] (2) Preparation of PLCL / ADM composite nanofibers: The PLCL / ADM composite nanofiber scaffold was cut into 3 mm × 3 mm square pieces and dissolved in deionized water. The pieces were homogenized in a high-speed homogenizer at a speed of 5000 rpm to 10000 rpm for 30 min to 60 min to obtain a nanofiber dispersion.

[0067] The nanofiber dispersion was then stored in a centrifuge tube, pre-frozen at -10°C for 1.5 hours, placed in a -80°C refrigerator overnight (i.e., super-freezing), and then freeze-dried (i.e., lyophilized) for 48 hours to obtain dry PLCL / ADM composite nanofibers, which were then sterilized with ethanol vapor and irradiated with ultraviolet light to obtain sterile PLCL / ADM composite nanofibers. The purpose of this step was to prepare the PLCL / ADM composite nanofibers for subsequent loading into GelMA.

[0068] Based on electrospinning technology and combined with high-speed homogenization technology to prepare PLCL / ADM composite nano-short fibers, the performance of PLCL / ADM composite nano-short fibers can be improved. As an important material for improving existing biomaterials, it enriches the existing structure and ultimately improves the wound repair performance of the wound repair agent containing it.

[0069] A specific embodiment of the present invention further provides an application of the above-mentioned PLCL / ADM composite nano-short fibers or the PLCL / ADM composite nano-short fibers prepared by the above-mentioned preparation method in a wound repair preparation.

[0070] A specific embodiment of the present invention further provides a wound repair preparation, which contains the above-mentioned PLCL / ADM composite nano-short fibers or the PLCL / ADM composite nano-short fibers prepared by the above-mentioned preparation method.

[0071] The present invention also provides a wound repair preparation comprising the following ingredients:

[0072] (1) the above-mentioned PLCL / ADM composite nanofibers or the PLCL / ADM composite nanofibers prepared by the above-mentioned preparation method;

[0073] (2) GelMA (gelatin methacrylate) solution.

[0074] In some specific embodiments, the mass volume ratio of the PLCL / ADM composite nanofibers and the GelMA solution is 1-6% (w / v), for example, 1%, 2%, 3%, 4%, 5%, or 6%; preferably, the degree of substitution of the GelMA is 30%-90%, and the molecular weight is 100kDa-200kDa, and the mechanical properties of the hydrogel after photocrosslinking are optimized by the degree of substitution and molecular weight.

[0075] In some embodiments, ADSCs are further included, and the volume ratio of the ADSCs to the GelMA solution is 0.5*10 4 —1*10 4 cells.

[0076] The present invention also provides a preparation process of the wound repair preparation, comprising the following steps:

[0077] Prepare a GelMA solution with a mass concentration of 5-30%;

[0078] The PLCL / ADM composite nanofibers are added to the GelMA solution and mixed to obtain a wound repair preparation.

[0079] Furthermore, the GelMA solution having a mass concentration of 5-30% comprises:

[0080] Prepare a 0.2% (w / v)-0.3% (w / v) initiator standard solution;

[0081] The GelMA was added to the initiator standard solution, and heated in a water bath at 60-80° C. in the dark for 25-35 min to dissolve the solution, with shaking during the process to allow the GelMA to fully infiltrate the solution, to prepare a GelMA solution with a mass concentration of 5-30%.

[0082] In some specific embodiments, the preparation process of the wound repair preparation comprises the following steps: taking PBS (phosphate buffered saline) and adding it to a brown bottle containing an initiator; heating and dissolving in a 50° C. water bath for 15 minutes, shaking several times during the heating; and preparing a 0.25% (w / v) initiator standard solution;

[0083] The required amount of GelMA (methacrylated gelatin) with an amino substitution degree of 30-90% and a molecular weight of 100-200 kDa is placed in a centrifuge tube. A standard initiator solution is added to the tube and shaken to fully soak the GelMA. The solution is then heated in a 70°C water bath in the dark for 30 minutes, with several shakes during the process. A 5-30% GelMA solution is prepared. The GelMA solution is then immediately sterilized with a 0.22 μm sterile syringe filter (to prevent gelation at low temperatures). Sterile PLCL / ADM composite nanofibers are added to the GelMA solution and gently pipetted to mix. The mixture is then drawn into a 1 mL syringe to obtain the PLCL / ADM composite nanofiber-loaded GelMA, the wound repair agent described above. The PLCL / ADM composite nanofibers can also improve the mechanical properties of the hydrogel.

[0084] In some specific embodiments, the mixture obtained by mixing is further comprised of resuspending the ADSCs cell pellet and beating evenly to prepare the wound repair preparation, wherein the mass volume ratio of the PLCL / ADM composite nanofibers and the GelMA solution is 1-6% (w / v), and the number volume ratio of the ADSCs to the GelMA solution is 0.5*10 per 1 mL of the GelMA solution.4 —1*10 4 cells.

[0085] Specifically, the ADSCs pellet was resuspended in injectable GelMA loaded with PLCL / ADM composite nanofibers that had not been cross-linked by UV light and then pipetted evenly to obtain injectable GelMA loaded with PLCL / ADM short fibers and ADSCs. The GelMA loaded with PLCL / ADM short nanofibers and ADSCs (adipose tissue-derived stem cells) prepared in this step is in a liquid state and has biological activity, effectively regulating the wound microenvironment and immune response.

[0086] This wound repair agent is in liquid form before use and can be applied to complex wounds, allowing the liquid gel to fit perfectly with the wound. It is then cured by ultraviolet light to form a gel state. It has the advantages of preventing the invasion of foreign microorganisms, ensuring moist healing of the wound, and avoiding tearing of the wound during traditional dressing changes.

[0087] The detailed specific embodiments are as follows:

[0088] 1. Preparation of PLCL / ADM composite nanofibers:

[0089] Example 1

[0090] This embodiment provides a PLCL / ADM composite nanofiber, comprising the following steps:

[0091] (1) Preparation of PLCL / ADM composite nanofiber scaffolds: A PLCL / ADM mixed spinning solution was prepared, wherein PLCL:ADM = 3:7, PLCL Mw ≈ 300,000 (LLA:CL = 50:50), dissolved in hexafluoroisopropanol (HFIP) at a mass volume ratio of 10%, and magnetically stirred overnight;

[0092] An ordinary flat plate was used as a receiving device to draw the PLCL / ADM mixed spinning solution into a syringe, which was connected to a 21-gauge stainless steel needle and a 13kV high voltage. Electrospinning was performed at a flow rate of 3 mL / h to prepare a PLCL / ADM composite nanofiber scaffold. Figure 1 As shown, Figure 1 (A) is an electron microscopy image of the PLCL / ADM composite nanofiber scaffold for morphological observation; Figure 1 (B) Fourier transform infrared spectrum of PLCL / ADM composite nanofiber scaffold, in which the two components are successfully doped;

[0093] (2) Preparation of PLCL / ADM composite nanofibers: The PLCL / ADM composite nanofiber scaffolds were cut into 3 mm × 3 mm square pieces and dissolved in deionized water. The pieces were homogenized at 10,000 rpm for 30 min using a high-speed homogenizer to obtain nanofiber dispersions.

[0094] The nanofiber dispersion was then stored in a centrifuge tube, pre-frozen at -10°C for 1.5 hours, placed in a -80°C refrigerator overnight, and then freeze-dried for 48 hours to obtain dry PLCL / ADM composite nanofibers. The fibers were then sterilized with ethanol vapor and irradiated with ultraviolet light to obtain sterile PLCL / ADM composite nanofibers with a diameter of 680 nm and an aspect ratio of 680:36.

[0095] Example 2

[0096] This embodiment provides a PLCL / ADM composite nanofiber, comprising the following steps:

[0097] (1) Preparation of PLCL / ADM composite nanofiber scaffolds: A PLCL / ADM mixed spinning solution was prepared, wherein PLCL:ADM = 7:3, PLCL Mw ≈ 300,000 (LLA:CL = 50:50), dissolved in hexafluoroisopropanol (HFIP) at a mass volume ratio of 15%, and magnetically stirred overnight;

[0098] Using a common flat plate as a receiving device, the PLCL / ADM mixed spinning solution was sucked into a syringe, connected to a 21-gauge stainless steel needle and connected to a 5kV high voltage, and electrospinning was performed at a flow rate of 1mL / h to produce a PLCL / ADM composite nanofiber scaffold.

[0099] (2) Preparation of PLCL / ADM composite nanofibers: The PLCL / ADM composite nanofiber scaffold was cut into 3 mm × 3 mm square pieces and dissolved in deionized water. The pieces were homogenized at 5000 rpm for 60 min using a high-speed homogenizer to obtain a nanofiber dispersion.

[0100] The nanofiber dispersion was then stored in a centrifuge tube, pre-frozen at -10°C for 1.5 hours, placed in a -90°C refrigerator for 10 hours, and then freeze-dried for 50 hours to obtain dry PLCL / ADM composite nanofibers. The fibers were then sterilized with ethanol vapor and irradiated with ultraviolet light to obtain sterile PLCL / ADM composite nanofibers with a diameter of 950 nm and an aspect ratio of 950:36.

[0101] Example 3

[0102] This embodiment provides a PLCL / ADM composite nanofiber, comprising the following steps:

[0103] (1) Preparation of PLCL / ADM composite nanofiber scaffolds: A PLCL / ADM mixed spinning solution was prepared, wherein PLCL:ADM=5:5, PLCL Mw≈300,000 (LLA:CL=50:50), dissolved in hexafluoroisopropanol (HFIP) at a mass volume ratio of 5%, and magnetically stirred overnight;

[0104] Using a common flat plate as a receiving device, the PLCL / ADM mixed spinning solution was sucked into a syringe, connected to a 21-gauge stainless steel needle and connected to a 15kV high voltage, and electrospinning was performed at a flow rate of 2mL / h to produce a PLCL / ADM composite nanofiber scaffold.

[0105] (2) Preparation of PLCL / ADM composite nanofibers: The PLCL / ADM composite nanofiber scaffolds were cut into 3 mm × 3 mm square pieces and dissolved in deionized water. The pieces were homogenized at 7000 rpm for 40 min using a high-speed homogenizer to obtain nanofiber dispersions.

[0106] The nanofiber dispersion was then stored in a centrifuge tube, pre-frozen at -10°C for 1.5 hours, placed in a -70°C refrigerator for 14 hours, and then freeze-dried for 45 hours to obtain dry PLCL / ADM composite nanofibers. The fibers were then sterilized with ethanol vapor and irradiated with ultraviolet light to obtain sterile PLCL / ADM composite nanofibers with a diameter of 800 nm and an aspect ratio of 800:40.

[0107] 2. Preparation of wound repair agent:

[0108] Example 4

[0109] This embodiment provides a preparation process of a wound repair preparation, comprising the following steps:

[0110] (1) Take PBS (phosphate buffered saline) and add it to a brown bottle containing the initiator (LAP phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt); heat and dissolve in a 50°C water bath for 15 minutes, shaking several times during the process; prepare a 0.25% (w / v) initiator standard solution;

[0111] (2) Place the required amount of GelMA (methacrylated gelatin) in a centrifuge tube, wherein the amino substitution degree of GelMA is 30-90% and the molecular weight is 100-200 kDa. Add the initiator standard solution to the above centrifuge tube, shake to fully soak the GelMA, and heat in a 70°C water bath in the dark for 30 minutes, shaking several times during the process to prepare a 20% GelMA solution.

[0112] (3) The GelMA solution was immediately sterilized using a 0.22 μm sterile syringe filter (to prevent low-temperature gelation); the sterile PLCL / ADM composite nanofibers prepared in Example 1 were added to the GelMA solution, gently blown and mixed using a pipette, and aspirated into a 1 mL syringe to obtain GelMA loaded with PLCL / ADM composite nanofibers, i.e., the above-mentioned wound repair agent, wherein the mass volume ratio of PLCL / ADM composite nanofibers to the GelMA solution was 3% (w / v).

[0113] Example 5

[0114] This embodiment provides a preparation process of a wound repair preparation, comprising the following steps:

[0115] (1) Take PBS (phosphate buffered saline) and add it to a brown bottle containing the initiator (LAP phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt); heat and dissolve in a 50°C water bath for 15 minutes, shaking several times during the process; prepare a 0.25% (w / v) initiator standard solution;

[0116] (2) Place the required amount of GelMA (methacrylated gelatin) in a centrifuge tube, wherein the amino substitution degree of GelMA is 30-90% and the molecular weight is 100-200 kDa. Add the initiator standard solution to the above centrifuge tube, shake to fully soak the GelMA, and heat in a 60°C water bath in the dark for 35 minutes, shaking several times during the process to prepare a 5% GelMA solution.

[0117] (3) The GelMA solution was immediately sterilized using a 0.22 μm sterile syringe filter (to prevent low-temperature gelation); the sterile PLCL / ADM composite nanofibers prepared in Example 2 were added to the GelMA solution, gently blown and mixed using a pipette, and the mixture was aspirated into a 1 mL syringe to obtain GelMA loaded with PLCL / ADM composite nanofibers, i.e., the above-mentioned wound repair agent, wherein the mass volume ratio of PLCL / ADM composite nanofibers to the GelMA solution was 1% (w / v).

[0118] Example 6

[0119] This embodiment provides a preparation process of a wound repair preparation, comprising the following steps:

[0120] (1) Take PBS (phosphate buffered saline) and add it to a brown bottle containing the initiator (LAP phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt); heat and dissolve in a 50°C water bath for 15 minutes, shaking several times during the process; prepare a 0.3% (w / v) initiator standard solution;

[0121] (2) Place the required amount of GelMA (methacrylated gelatin) in a centrifuge tube, wherein the amino substitution degree of GelMA is 30-90% and the molecular weight is 100-200 kDa. Add the initiator standard solution to the above centrifuge tube, shake to fully soak the GelMA, and heat in a water bath at 80°C in the dark for 25 minutes, shaking several times during the process to prepare a 30% GelMA solution.

[0122] (3) The GelMA solution was immediately sterilized using a 0.22 μm sterile syringe filter (to prevent low-temperature gelation); the sterile PLCL / ADM composite nanofibers prepared in Example 1 were added to the GelMA solution, gently blown and mixed using a pipette, and the mixture was aspirated into a 1 mL syringe to obtain GelMA loaded with PLCL / ADM composite nanofibers, i.e., the above-mentioned wound repair agent, wherein the mass volume ratio of PLCL / ADM composite nanofibers to the GelMA solution was 6% (w / v).

[0123] Example 7

[0124] This embodiment provides a preparation process of a wound repair preparation, comprising the following steps:

[0125] The wound repair agent prepared in Example 4 without UV cross-linking was resuspended with the ADSCs cell pellet and pipetted evenly to prepare a wound repair preparation containing ADSCs (i.e., a bioactive hydrogel dressing), wherein the mass volume ratio of PLCL / ADM composite nanofibers to the GelMA solution was 3% (w / v), and the number volume ratio of ADSCs to the GelMA solution was 0.8*10 4 cells; such as Figure 3 As shown, Figure 3 (A) is a photo of the object before light curing; Figure 3 (B) Photo of the glue formed after light-curing and cross-linking. It can be attached to the bottom of the dish without slipping and can fit the wound surface.

[0126] Example 8

[0127] This embodiment provides a preparation process of a wound repair preparation, comprising the following steps:

[0128] The wound repair agent prepared in Example 5 without UV cross-linking was resuspended with the ADSCs cell pellet and pipetted evenly to prepare a wound repair preparation containing ADSCs (i.e., a bioactive hydrogel dressing), wherein the mass volume ratio of PLCL / ADM composite nanofibers to the GelMA solution was 1% (w / v), and the number volume ratio of ADSCs to the GelMA solution was 1*10 per 1 mL of the GelMA solution. 4 cells.

[0129] Comparative Example 1

[0130] The only difference from Example 1 is that ADM is not added in this comparative example.

[0131] Comparative Example 2

[0132] The only difference from Example 1 is that PCL is used instead of PLCL in this comparative example.

[0133] Comparative Example 3

[0134] The only difference from Example 1 is that in this comparative example, PLCL:ADM=2:7. When used in wound repair, the injectability of the hydrogel is lower than that of Example 1, and the ability to gradually release various effective substances to promote collagen production and accelerate wound healing is also lower than that of Example 1.

[0135] Comparative Example 4

[0136] The only difference from Example 1 is that in this comparative example, PLCL:ADM=7:2. When used in wound repair, the injectability of the hydrogel is lower than that of Example 1, and the ability to gradually release various effective substances to promote collagen production and accelerate wound healing is also lower than that of Example 1.

[0137] Test example:

[0138] (1) Cell proliferation experiment of fibroblasts seeded on the PLCL / ADM composite nanofiber scaffold (i.e., nanofiber membrane) and blank glass slide prepared in Example 1: The prepared PLCL / ADM composite nanofiber scaffold was punched into a 1 cm diameter disc as the experimental group, and a 1 cm diameter disc as the control group. The 24-well plates were placed and sterilized with ethanol vapor and ultraviolet irradiation, and 0.8 × 10 ADSCs were seeded in each well. 4 On days 1, 3, 5, and 7, the proliferation activity of fibroblasts was measured using a cell counting kit (CCK-8). A culture medium containing 10% CCK-8 reagent was added to each well and incubated in a 37°C, 5% CO2 incubator for 2 hours. The absorbance (OD) of the culture medium containing CCK-8 reagent in each well was then measured at 450 nm using a multifunctional microplate reader. A higher OD value indicates a greater number of cells. This was used to evaluate the biocompatibility of the PLCL / ADM composite nanofiber scaffold.

[0139] like Figure 2 As shown, it shows that the PLCL / ADM composite nanofiber scaffold has good biocompatibility, and the number of cell proliferation is much higher than that of the blank wave plate over time.

[0140] (2) The same amount of ADSCs was loaded onto GelMA and the wound repair agent prepared in Example 4, and the cell proliferation of ADSCs in each was observed. Figure 4 As shown, it is proved that the wound repair agent prepared in Example 4 effectively promotes the cell proliferation of ADSCs.

[0141] (3) Animal experiment: A full-thickness skin defect model was established on the back of rats.

[0142] Experimental group: The wound repair preparation containing ADSCs prepared in Example 7 (i.e., GelMA loaded with PLCL / ADM composite nanofibers and ADSCs, or the novel dressing) was injected into the wound surface on the back of rats to evenly cover the wound surface. The wound was then photocrosslinked with 405 nm ultraviolet light for 20 seconds to form a gel.

[0143] Control group: oil gauze (also known as gauze) covered the wound surface;

[0144] Blank group: no treatment;

[0145] like Figure 5 As shown, Figure 5 (A) Schematic diagram showing the wound healing process in a rat full-thickness skin defect model; Figure 5 (B) Bar graph of wound healing rate calculation, which proves that the wound repair preparation containing ADSCs promotes wound healing significantly better than other groups.

[0146] like Figure 6 As shown, Figure 6 (A) HE staining of wound healing tissue on day 7; Figure 6 (B) Histogram of the epidermal thickness of the healed wound surface; this demonstrates that the new dressing promotes wound healing and epidermalization better than other groups.

[0147] like Figure 7 As shown, Figure 7 (A) HE staining of wound healing tissue on day 7; Figure 7 (B) Histogram of collagen content in healing wounds; this demonstrates that the new dressing promotes wound healing and collagen regeneration to a greater extent than other groups.

[0148] (4) Test of hydrogel mechanical properties: GelMA was used as the control group and GelMA loaded with PLCL / ADM composite nanofibers prepared in Example 4 was used as the experimental group. The shear properties before photocrosslinking and the stress-strain changes after photocrosslinking were measured respectively. The corresponding test results are shown in Figure 2. Figure 8As shown, the left figure shows the shear thinning of GelMA before photocrosslinking, proving that the addition of short fibers does not affect the injectability of GelMA. The right figure shows the compressive stress / strain diagram of GelMA after photocrosslinking, proving that the mechanical properties of GelMA are improved after the addition of short fibers.

[0149] (5) Fibroblasts were seeded on the PLCL / ADM composite nanofiber scaffold (also known as nanofiber membrane) prepared in Example 1, a blank glass slide, and a PLCL nanofiber membrane (Comparative Example 1) Cell proliferation experiment: The prepared PLCL / ADM composite nanofiber scaffold was punched into a 1 cm diameter circular sheet as the experimental group, and a 1 cm diameter circular sheet and a PLCL nanofiber membrane were used as the control group. The 24-well plates were placed and sterilized with ethanol vapor and ultraviolet irradiation, and 0.8 × 10 ADSCs were seeded in each well. 4 On days 1, 3, 5, and 7, the proliferation activity of fibroblasts was measured using a cell counting kit (CCK-8). A culture medium containing 10% CCK-8 reagent was added to each well and incubated in a 37°C, 5% CO2 incubator for 2 hours. The absorbance (OD) of the culture medium containing CCK-8 reagent in each well was then measured at 450 nm using a multifunctional microplate reader. A higher OD value indicates a greater number of cells. This was used to evaluate the biocompatibility of the PLCL / ADM composite nanofiber scaffold.

[0150] like Figure 9 As shown, it shows that the PLCL / ADM composite nanofiber scaffold has good biocompatibility, and the number of cell proliferation is much higher than that of the blank wave plate and PLCL nanofiber membrane over time.

[0151] (6) Degradation performance test: The PCL / ADM nanofiber scaffold prepared in Comparative Example 2 and the PLCL / ADM composite nanofiber scaffold prepared in Example 1 were immersed in PBS at 37°C. After drying the residual surface moisture on the 7th and 14th days, the degradation rate was measured by weighing. Figure 10 As shown, it was found that the PCL / ADM nanofiber scaffold prepared in Comparative Example 2 degraded slowly, which was not conducive to the rapid release of effective substances to promote the improvement of wound repair speed during the wound repair proliferation period.

[0152] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A wound repair preparation, characterized in that: Includes the following ingredients: (1) PLCL / ADM composite nanofibers, wherein the mass ratio of PLCL to ADM is (3-7): (3-7); The diameter of the PLCL / ADM composite nanofibers is (680 ± 270) nm; The aspect ratio of the PLCL / ADM composite nanofiber is (950-410): (67-5); The preparation method of the PLCL / ADM composite nanofiber comprises the following steps: Preparation of PLCL / ADM composite nanofibers; homogenizing the PLCL / ADM composite nanofibers to obtain a nanofiber dispersion; The nano-short fiber dispersion is freeze-dried to obtain PLCL / ADM composite nano-short fibers, wherein the freeze-drying comprises first freezing the nano-short fiber dispersion at -30°C to -10°C for 0.5 to 2 hours, then superfreezing at -90°C to 70°C, and then freeze-drying for 45 to 50 hours; (2) a GelMA solution, wherein the GelMA has a degree of substitution of 30% to 90% and a molecular weight of 100 kDa to 200 kDa; The mass volume ratio of the PLCL / ADM composite nano short fibers to the GelMA solution is 1-6% (w / v).

2. The wound repair preparation according to claim 1, characterized in that ADSCs are also included, and the volume ratio of the ADSCs to the GelMA solution is 0.5 per 1 mL of the GelMA solution. 10 4 —1 10 4 cells.

3. The preparation process of the wound repair preparation according to any one of claims 1-2, characterized in that: The steps include: Prepare a GelMA solution with a mass concentration of 5-30%; The PLCL / ADM composite nanofibers are added to the GelMA solution and mixed to obtain a wound repair preparation.

4. The preparation process according to claim 3, characterized in that The invention also includes resuspending the ADSCs cell sediment by mixing the obtained mixed solution, and blowing it evenly to prepare the wound repair preparation, wherein the mass volume ratio of the PLCL / ADM composite nanofibers and the GelMA solution is 1-6% (w / v), and the number volume ratio of the ADSCs to the GelMA solution is 0.5 per 1 mL of the GelMA solution. 10 4 —1 10 4 cells; and / or, The GelMA solution configured to have a mass concentration of 5-30% comprises: Prepare a 0.2% (w / v)-0.3% (w / v) initiator standard solution; The GelMA was added to the initiator standard solution, and heated in a water bath at 60-80° C. in the dark for 25-35 min to dissolve the solution, with shaking to allow the GelMA to fully infiltrate the solution, to prepare a GelMA solution with a mass concentration of 5-30%.

5. The preparation process according to claim 3, characterized in that: The preparation method of the PLCL / ADM composite nano short fibers comprises the following steps: Preparation of PLCL / ADM composite nanofibers; homogenizing the PLCL / ADM composite nanofibers to obtain a nanofiber dispersion; The nano-short fiber dispersion is freeze-dried to prepare PLCL / ADM composite nano-short fibers.

6. The preparation process according to claim 5, characterized in that: The homogenization speed is 5000 rpm-10000 rpm, and the time is 30 min-60 min; The nanofiber dispersion is first frozen at -30°C to -10°C for 0.5h to 2h, then super-frozen at -90°C to 70°C, and then freeze-dried for 45h to 50h.

7. The preparation process according to claim 5, characterized in that: The preparation of PLCL / ADM composite nanofibers comprises the following steps: Dissolve PLCL and ADM in a solvent to prepare a PLCL / ADM mixed spinning solution: The PLCL / ADM mixed spinning solution is subjected to electrostatic spinning to prepare PLCL / ADM composite nanofibers.

8. The preparation process according to claim 7, characterized in that: The step of dissolving PLCL and ADM in a solvent to prepare a PLCL / ADM mixed spinning solution comprises: dissolving PLCL and ADM in a mass ratio of (3-7): (3-7) in hexafluoroisopropanol at a mass volume ratio of 5-15% to prepare a PLCL / ADM mixed spinning solution; and / or, the electrospinning voltage is 5kV-15 kV, and the flow rate is 1 mL / h-3 mL / h.

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