Wound dressing for burns and scalds, its preparation method and its application

Abstract

The present invention discloses a wound dressing for burn and scald, its preparation method and its application, belonging to the field of medical dressings. The technical solution includes dissolving polycaprolactone and polylysine in medical acetone, and stirring evenly with a magnetic stirrer to obtain the shell solution; activating platelet-rich plasma with polyvinyl alcohol and calcium chloride to obtain the core solution; mixing the shell solution and the core solution according to a volume ratio of 1:2, and using a coaxial electrospinning device for electrospinning to obtain platelet-rich plasma@polycaprolactone-polylysine core-shell nanofibers. The present invention is applied to the treatment of burn and scald. When using PRP to treat burn and scald, directly applying it to the affected area will cause the growth factors to be quickly decomposed by enzymes in the wound microenvironment, resulting in poor treatment effects. The present invention can encapsulate PRP into nanofibers, effectively avoiding the problem of solvent miscibility in electrospinning, and has excellent antibacterial properties and tissue regeneration promotion characteristics.

Description

Technical Field

[0001] The present invention belongs to the field of medical dressings, and particularly relates to a wound dressing for burns and scalds, a preparation method thereof, and an application thereof. Background Art

[0002] After burns and scalds, the skin is damaged, the supply of enzymes and growth factors is blocked, and without the protection of the skin, it is extremely vulnerable to bacterial infection, resulting in slow recovery of burns and scalds treatment. Different from other skin damages, such as scratches, stabs, and cuts, burns and scalds are injuries caused by the skin encountering local excessive heat. Therefore, at the first moment of burns and scalds, if cold water is used to flush the wound, symptoms such as swelling and blisters on the wound surface can be relieved. However, currently reported dressings for burns and scalds generally only focus on the later repair process and ignore the first-time cooling first aid. This may be because it is considered that in cities, there is easily available pure water that can be used for first aid. However, in the wild environment, there is a lack of clean water, and using dirty water to flush the wound will cause serious bacterial infection.

[0003] Hospitals generally use gauze and burn and scald ointments to treat burns and scalds. However, the gauze has too high porosity, too large pore size, and poor skin adhesion, which is not conducive to preventing the invasion of external bacteria. And the ointment, as an exogenous drug, has relatively single components and often needs to be used in combination, and the treatment effect varies from person to person. Autologous PRP (Platelet Rich Plasma, platelet-rich plasma) not only contains rich growth factors, which can promote angiogenesis and cell proliferation, but also because it is autologous, the component ratio of each growth factor in PRP varies slightly from person to person, which can achieve personalized treatment and avoid allergic reactions of the body.

[0004] However, when PRP is directly applied to the affected area, after PRP is activated, growth factors are released. However, because the growth factors will be rapidly decomposed by enzymes in the wound microenvironment, it is difficult to continuously maintain a high concentration. Summary of the Invention

[0005] Aiming at the deficiencies of the existing technology, the technical problem to be solved by the present invention is to overcome the problem that when using PRP to treat burns and scalds, directly applying it to the affected area results in poor treatment effect due to the rapid decomposition of growth factors by enzymes in the wound microenvironment. The present invention provides a wound dressing for burns and scalds, a preparation method thereof, and an application thereof, which can encapsulate PRP into nanofibers, effectively avoid the problem of solvent miscibility in electrospinning, and have excellent antibacterial properties and tissue regeneration promotion characteristics.

[0006] To solve the above technical problem, the technical solution adopted by the present invention is as follows:

[0007] On the one hand, the present invention provides a preparation method of a wound dressing for burns and scalds, including:

[0008] The preparation step of the shell solution includes dissolving polycaprolactone and polylysine in medical acetone and stirring evenly with a magnetic stirrer to obtain the shell solution;

[0009] The preparation step of the core layer solution includes activating platelet-rich plasma with polyvinyl alcohol and calcium chloride to obtain the core layer solution;

[0010] The preparation step of platelet-rich plasma@polycaprolactone-polylysine core-shell nanofibers includes mixing according to the volume ratio of the shell solution to the core layer solution of 1:2 and performing electrospinning using a coaxial electrospinning device to obtain the platelet-rich plasma@polycaprolactone-polylysine core-shell nanofibers.

[0011] Preferably, the preparation step of the shell solution specifically includes:

[0012] Dissolve 2 g of polycaprolactone and 0.4 g of polylysine in 10 g of medical acetone and stir with a magnetic stirrer for 12 h to obtain the shell solution.

[0013] Preferably, the preparation step of the core layer solution specifically includes:

[0014] Activate platelet-rich plasma with 5% (mass concentration) of polyvinyl alcohol and calcium chloride to obtain the core layer solution.

[0015] Preferably, the conditions for electrospinning using the coaxial electrospinning device are: the diameter of the electrospinning nozzle is 0.4 mm, the electrospinning voltage is 10 kV, and the distance between the electrospinning nozzle and the collector is 10 cm.

[0016] Preferably, the platelet-rich plasma@polycaprolactone-polylysine core-shell nanofibers are collected on an aluminum foil connected to the negative electrode.

[0017] Preferably, the coaxial electrospinning device is provided with a test tube, and a semiconductor refrigerating sheet is provided on the surface of the test tube.

[0018] Preferably, the coaxial electrospinning device uses two alkaline batteries as the power supply and is equipped with a high-voltage converter to convert 3 V to 10 kV.

[0019] On the other hand, the present invention provides a wound dressing for burns and scalds, which is prepared by the preparation method of the wound dressing for burns and scalds described in any one of the above technical solutions.

[0020] The present invention also provides an application of the wound dressing for burns and scalds according to the above in burns and scalds.

[0021] Preferably, the coaxial electrospinning equipment is used to spin the mixed solution of the shell layer solution and the core layer solution, and the obtained platelet-rich plasma@polycaprolactone-polylysine core-shell nanofibers are directly deposited on the skin to achieve the treatment of burns and scalds.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] The present invention provides a preparation method for a wound dressing for burns and scalds, which can encapsulate PRP into nanofibers, effectively avoid the problem of solvent miscibility in electrospinning, and the prepared wound dressing for burns and scalds has excellent antibacterial properties and tissue regeneration promotion characteristics. Description of the Drawings

[0024] Figure 1 SEM image of the PRP-containing composite nanofiber membrane provided by the embodiment of the present invention;

[0025] Figure 2 TEM image of the PRP-containing composite nanofiber membrane provided by the embodiment of the present invention;

[0026] Figure 3 Fibers labeled with calcein provided by the embodiment of the present invention;

[0027] Figure 4 Fibers labeled with rhodamine B provided by the embodiment of the present invention;

[0028] Figure 5 Schematic diagram of the structure of the PRP-containing composite nanofiber membrane under a confocal microscope provided by the embodiment of the present invention;

[0029] Figure 6 FTIR spectrum of the PRP-containing composite nanofiber membrane provided by the embodiment of the present invention;

[0030] Figure 7 Schematic diagram of the BET result of the PRP-containing composite nanofiber membrane provided by the embodiment of the present invention;

[0031] Figure 8 Water contact angles of different components;

[0032] Figure 9 Cumulative release curves of different growth factors;

[0033] Figure 10 Comparison diagram of the fiber membrane before and after cooling taken by an infrared thermal imager;

[0034] Figure 11 Comparison diagram of the cooling curves of the experimental group and the control group;

[0035] Figure 12 Schematic diagram of cooling;

[0036] Figure 13 HE staining of the burn and scald area of the rat without cooling treatment;

[0037] Figure 14 HE staining of the burn and scald area of the rat with in-situ electrospinning refrigeration;

[0038] Figure 15 Growth of Escherichia coli and Staphylococcus aureus after incubation on an agar plate for 24 hours;

[0039] Figure 16 SEM control images before and after the rupture of Escherichia coli;

[0040] Figure 17 SEM images before and after the rupture of Staphylococcus aureus;

[0041] Figure 18 Column chart of cell proliferation;

[0042] Figure 19 Comparison of wound healing among different control groups;

[0043] Figure 20 Column chart plotted according to the wound area of the rat among different control groups;

[0044] Figure 21 HE staining images of deep second-degree burn and scald wounds treated in different ways, with the bar representing 200 μm;

[0045] Figure 22 Masson staining images of deep second-degree burn and scald wounds treated in different ways, with the bar representing 200 μm. Specific implementation manner

[0046] The technical solutions in the specific embodiments of the present invention will be described in detail and completely below. Obviously, the described embodiments are only partial specific implementation manners of the general technical solution of the present invention, rather than all implementation manners. Based on the general concept of the present invention, all other embodiments obtained by those of ordinary skill in the art fall within the protection scope of the present invention.

[0047] One aspect of the present invention provides a preparation method of a wound dressing for burn and scald, including:

[0048] A step of preparing a shell layer solution, including dissolving polycaprolactone and polylysine in medical acetone and stirring evenly with a magnetic stirrer to obtain the shell layer solution;

[0049] A step of preparing a core layer solution, including activating platelet-rich plasma with polyvinyl alcohol and calcium chloride to obtain the core layer solution;

[0050] Preparation steps of platelet-rich plasma@polycaprolactone-polylysine core-shell nanofibers include mixing according to the volume ratio of the shell solution to the core solution of 1:2, and electrospinning using a coaxial electrospinning device to obtain the platelet-rich plasma@polycaprolactone-polylysine core-shell nanofibers.

[0051] The present invention provides a preparation method of a wound dressing for burns and scalds. Among them, autologous PRP (Platelet Rich Plasma, platelet-rich plasma) not only contains abundant growth factors that can promote angiogenesis and cell proliferation, but also because it is autologous, the component ratio of each growth factor in PRP varies slightly from person to person, enabling personalized treatment and avoiding allergic reactions of the body. However, when PRP is directly applied to the affected area, after being activated, PRP releases growth factors. However, due to the fact that growth factors will be rapidly decomposed by enzymes in the wound microenvironment, it is difficult to continuously maintain a high concentration. Encapsulating PRP into nanofibers can avoid this shortcoming. Considering that PRP is water-soluble while most biodegradable materials are hydrophobic materials, forming a core-shell structure fiber with PRP and fibers can avoid the problem of solvent miscibility in electrospinning.

[0052] In a preferred embodiment, the preparation steps of the shell solution specifically include: dissolving 2 g of polycaprolactone and 0.4 g of polylysine in 10 g of medical acetone and stirring for 12 h with a magnetic stirrer to obtain the shell solution.

[0053] In a preferred embodiment, the preparation steps of the core solution specifically include: activating platelet-rich plasma with 5% (mass concentration) polyvinyl alcohol and calcium chloride to obtain the core solution.

[0054] In a preferred embodiment, the conditions for electrospinning using the coaxial electrospinning device are: the diameter of the electrospinning nozzle is 0.4 mm, the electrospinning voltage is 10 kV, and the distance between the electrospinning nozzle and the collector is 10 cm.

[0055] In a preferred embodiment, the platelet-rich plasma@polycaprolactone-polylysine core-shell nanofibers are collected on an aluminum foil connected to the negative electrode.

[0056] In a preferred embodiment, the coaxial electrospinning device is provided with a test tube, and a semiconductor refrigerating sheet is disposed on the surface of the test tube. Preferably, the coaxial electrospinning device is a handheld coaxial electrospinning device. By providing the semiconductor refrigerating sheet, the fibers can be synchronously turned into ice-cold fibers during the electrospinning process and directly deposited on the skin outdoors. Through a two-fold cooling method, the residual heat at the burn wound is taken away to achieve first-aid at the first time. In addition, these fibers exhibit excellent antibacterial properties and tissue regeneration promoting properties. Moreover, since the handheld coaxial electrospinning can achieve in-situ deposition, it can further enhance the antibacterial properties and the effect of growth factors in promoting wound healing. This strategy of integrating cooling, antibacterial, and wound healing acceleration shortens the burn recovery time from 21 days to 14 days.

[0057] In a preferred embodiment, the coaxial electrospinning device uses two alkaline batteries as a power source and is equipped with a high-voltage converter to convert 3V into 10kV.

[0058] On the other hand, the present invention provides a wound dressing for burns, which is prepared by the preparation method of the wound dressing for burns according to any of the above technical solutions. The wound dressing has excellent antibacterial properties and tissue regeneration promoting properties. Its strategy of integrating cooling, antibacterial, and wound healing acceleration shortens the burn recovery time from 21 days to 14 days.

[0059] The present invention also provides an application of the above-mentioned wound dressing for burns in the treatment of burns.

[0060] In a preferred embodiment, the coaxial electrospinning device is used to electrospin a mixture of the shell solution and the core solution, and the obtained platelet-rich plasma@polycaprolactone-polylysine core-shell nanofibers are directly deposited on the skin to achieve the treatment of burns. By providing the semiconductor refrigerating sheet, the fibers can be synchronously turned into ice-cold fibers during the electrospinning process and directly deposited on the skin outdoors. Through a two-fold cooling method, the residual heat at the burn wound is taken away to achieve first-aid at the first time. In addition, these fibers exhibit excellent antibacterial properties and tissue regeneration promoting properties. Moreover, since the handheld coaxial electrospinning can achieve in-situ deposition, it can further enhance the antibacterial properties and the effect of growth factors in promoting wound healing. This strategy of integrating cooling, antibacterial, and wound healing acceleration shortens the burn recovery time from 21 days to 14 days.

[0061] In order to more clearly and detailedly introduce the wound dressing for burns, its preparation method, and its application provided by the embodiments of the present invention, the following will be described in conjunction with specific embodiments.

[0062] Example 1

[0063] In order to prepare the PRP-containing coaxial electrospun fiber membrane, we dissolved 2g PCL and 0.4g ε-PL in 10g medical acetone and stirred them in a magnetic stirrer for 12 hours to obtain the shell solution for coaxial electrospinning. We dissolved 5% PVA (5% PVA is 0.5g polyvinyl alcohol dissolved in 10g water) and CaCl 2 The activated PRP was tested for blood routine. As the core layer solution (ratio of 1:2), PCL, PCL+ε-PL, PCL+PRP+ε-PL fiber membranes were prepared using conventional electrospinning equipment. The voltage of the above groups was 17.6KV, and the flow rates of the core layer and shell layer solutions were 0.5ml / h and 1.2ml / h, respectively. The distance between the needle and the receiving electrode was about 15cm, and all spinning processes were carried out at 25℃ and 50% humidity. In the in-situ electrospinning PCL+PRP+ε-PL group, the spinning nozzle diameter was 0.4mm, the spinning voltage was 10kV, and the distance between the spinning nozzle and the collector was 10cm. The nanofibers were collected on an aluminum foil connected to the negative electrode.

[0064] Regarding raw materials, polycaprolactone (PCL), poly-lysine (ε-PL) and polyvinyl alcohol (PVA) were purchased from Aladdin, and medical acetone was from Zhongshan Yiya Biotechnology Co., Ltd. Calcium chloride (CaCl 2 ) was purchased from Sinopharm Chemical Reagent Co., Ltd., ready-to-use nutrient agar medium was purchased from Jiangmen Kailin Trading Co., Ltd., Escherichia coli, Staphylococcus aureus, fibroblasts and growth medium were provided by the Affiliated Hospital of Qingdao University Medical College, high glucose medium (DMEM) was purchased from Beijing Solebow Technology Co., Ltd., fetal bovine serum (FBS) and trypsin were purchased from BI, CCK-8 reagent was purchased from bio sharp, and cytoskeleton dye (Phalloidin-iflour 488reagent) was purchased from abeam. All chemicals and solvents were of reagent grade and used without further purification.

[0065] Regarding the device, the cooling coaxial electrospinning device used uses two 5ml syringes placed in the test tube compartment, an aluminum conical auxiliary electrode is fixed at the end of the coaxial needle, and plastic tubes that can pass the solution are connected at both ends of the needle. Finally, semiconductor refrigeration sheets are placed on the surface of the two test tubes. The device uses two alkaline batteries (1cm in diameter, 4.5cm in height, 1.5v, Nanfu battery LR03) as power supply, and is equipped with a high-voltage converter (3V to 10kV), high-voltage power supply, and the distances between the needle and the receiving electrode are: 10kv, 10cm respectively.

[0066] Performance Testing

[0067] 1. Release of growth factors from fiber membranes

[0068] Prepare a 24-well plate containing phosphate-buffered saline. Punch the fiber membranes in each group and place them in it. After incubating for one month, replace the original PBS with fresh PBS. Use an enzyme-linked immunosorbent assay kit to measure the release of PDGF-BB, VEGF, and TGF-β released at each time point. Finally, monitor the absorbance at 450 nm with an enzyme-labeled instrument and plot the standard curve of the average concentration of growth factors.

[0069] 2. Cooling experiment

[0070] To examine the effect of cooling on burns, we used an infrared thermal imager to compare the cooled fiber membranes with those without cooling treatment. First, heat the platform to 80 °C. In the control group, no sample is placed on the platform, and then cooling is carried out. Record the temperature every 10 seconds, from 0 seconds to 300 seconds; in the experimental group, place the cooled fiber membrane on the heated platform and record the temperature by taking pictures in the same way as above.

[0071] 3. In vitro antibacterial experiment

[0072] We evaluated the antibacterial effect by the disc agar diffusion method. First, in a sterile environment, inoculate the suspensions of Escherichia coli and Staphylococcus aureus with about 1×108 cells onto a ready-to-use nutrient agar medium (9 cm×9 cm). Cut the fiber membranes in several experimental groups into fiber pads with a radius of 1 cm, and then place the fiber pads at the center of the 9 cm×9 cm agar medium coated with bacteria. Culture in a constant temperature and pressure incubator at 30 °C for 18 hours.

[0073] 4. Biocompatibility test

[0074] 4.1 Culture of human foreskin fibroblasts

[0075] The whole process is carried out under sterile operation and completed in a sterile cell room.

[0076] In the first step, disinfect and soak the excised whole foreskin tissue with alcohol on a laminar flow bench for 5 minutes, then rinse it 3 times with sterile distilled water, and finally rinse it with 5% double antibody dissolved in PBS. In the second step, use a sterilized surgical scissors to remove the outer epidermis and subcutaneous tissue, and only retain the dermis layer. Cut the remaining white tissue into rectangular tissue blocks about 5*5 mm in size, evenly spread them on the bottom of the culture dish, add an appropriate amount of complete medium with 15% FBS, ensure that the medium covers the tissue blocks and the bottom of the tissue blocks is closely attached to the bottom of the culture dish, and transfer the culture dish into a cell constant temperature incubator for culture. Replace the cell culture medium every 2 days. After about 5-7 days, single fibroblasts can be observed crawling out around the tissue blocks. Generally, after about 8-14 days, a large number of primary fibroblasts can be seen aggregating around the tissue blocks, and preliminary passage can be carried out.

[0077] 4.2 Cytotoxicity test

[0078] Since the PRP-containing coaxial electrospun fiber membrane is ultimately used in the human body, it is required to be non-toxic. Therefore, fibroblasts were seeded on different glass slides in 4 groups and a control group, and their cellular responses were detected. After alcohol disinfection and ultraviolet irradiation of the nanofiber membrane, the cell metabolic activity was measured using CCK-8 (Figure S2). All experimental procedures were carried out under a sterile laminar flow hood. This experiment was divided into the above 5 groups, including the traditional electrospinning group of PCL, PCL + ε-PL group, PCL + PRP + ε-PL group, and in-situ PCL + ε-PL + PRP group. There were 4 replicate wells in each group. The above 5 groups of composite nanofiber membranes and the control group of empty glass slides were placed in a 24-well plate, washed once with PBS, washed three times with serum-free medium, then 100 μl of complete medium was added, and then placed in a 37 °C incubator.

[0079] Next, fibroblasts with a confluence of 90% were taken out of the incubator, rinsed 3 times with PBS, then 200 μl of trypsin was added and placed in the incubator to digest the cells. After 3 minutes, it was taken out and the digestion was terminated with 1 ml of complete medium (DMEM high glucose hyclone, 10% fetal bovine serum, 1% double antibody). The cells were repeatedly pipetted until they detached and then placed in a 1.5 ml EP tube and centrifuged at 900 rmp for 5 min. The supernatant was discarded, and the cells were resuspended in 1 ml of complete medium. 10 μl of the cell suspension was taken for counting, and the number of cells obtained was 80×104 cells / ml. The number of cells seeded in each well was approximately 105 cells / ml. After preparing the required cell suspension, 100 μl of the cell suspension was pipetted into each well using a pipette gun. After 2 hours, the medium was supplemented to 700 μl. The cell proliferation numbers at 4 hours, 12 hours, 1, 3, and 5 days were measured respectively. After removing the old medium, the cells were rinsed once with PBS, and complete medium containing 10% CCK-8 reagent (400 μl per well) was added. Note that the addition of the liquid should be carried out in the dark. Incubate in a humidified atmosphere of 5% CO 2 at 37 °C for 2 h. The liquid in the 2-well plate (100 μL) was added to a 96-well plate, with 3 replicate wells in each group, wrapped with tin foil, and the absorbance at 450 nm was monitored using a microplate reader.

[0080] 4.3 Cell Staining

[0081] The attachment and morphology of human fibroblasts were determined by double-label fluorescence staining of the actin cytoskeleton and nucleus of fibroblasts. First, the cells were co-cultured with different groups of fibrous membranes and glass slides to determine whether the materials exhibited any cytotoxicity. The composite nanofiber membranes were cut into the size of 24-well plates, fixed in 24-well plates with steel rings, the lids were opened and placed in a glass drying jar, and after being disinfected with 75% alcohol vapor for six hours and ultraviolet (UV) irradiation for one hour, they were used for experiments. Then all samples were washed with PBS and serum-free cell culture medium. Human dermal fibroblasts were inoculated in 24-well plates at a density of 1×104 cells / well, taken out after co-culturing for 12 hours and 3 days respectively, and fixed with 3% paraformaldehyde for 20 min. Then the cell membranes were permeabilized with 0.1% TritonX-100 solution for 5 min and rinsed three times with PBS. After blocking with 1% BSA solution for one hour, they were washed three times with PBS. Finally, the cells were stained with DAPI and FITC-Phalloidin (abcam, Shanghai, China), and these images were taken by an upright fluorescence microscope (Nikon A1MP, Japan).

[0082] 5. In vivo animal experiments

[0083] 5.1 Establishment of animal models

[0084] All SPF-grade male SD rats were purchased from Nanjing Junke Bioengineering Co., Ltd., each weighing about 200 g, and all rats were intervened and treated according to the guidelines of the Animal Laboratory Supervision and Administration Committee of the Ministry of Health of the Chinese government. First, a 10% chloral hydrate solution was prepared, and the rats were anesthetized by intraperitoneal injection at a dose of 300 mg / kg. Then the backs of the rats were depilated, rinsed clean, disinfected with alcohol, and two symmetrical scalded circular areas with a diameter of 1.4 cm were made on both sides of the spine. Finally, the surface skin was disinfected with alcohol.

[0085] 5.2 Animal experiments

[0086] To test the curative effect, 40 SD rats were randomly divided into 5 groups. Appropriate-shaped PCL, PCL + ε-PL, and PCL + ε-PL + PRP fiber scaffolds were cut according to the shape of the skin scald wounds. In the in-situ group, the PCL + PRP + ε-PL composite nanofiber membrane was directly deposited in situ on the wound surface of the rats' back skin using a handheld electrospinning device. The wounds of each group were covered with gauze and fixed with silk sutures. To avoid interference between animals, they were raised individually in cages. After the scalded rats' wounds were covered with scaffolds or healed naturally, photos were taken and the healing conditions were observed on days 1, 3, 5, 7, 9, 14, and 21.

[0087] 6. Histological analysis

[0088] Subsequent histological analysis was performed. After taking frozen sections of the newly formed tissue, methods such as HE staining and Masson staining were used to observe the healing process of the rat skin wound surface (including wound inflammatory response, fibroblast proliferation, collagen secretion and typing, epidermal layer differentiation, etc.). The scalded tissues of SD rats on the 1st day, 7th day, 14th day, and 21st day were fixed on glass slides for hematoxylin-eosin (H&E) and Masson staining. HE and Masson staining were used to evaluate the number of bacteria around the wound and the progress of wound recovery, respectively. The histopathological changes of the wound were observed under a microscope.

[0089] 7. Characterization

[0090] The morphology of the fibers was analyzed using a scanning electron microscope (SEM, EV0MA25 / LS25) and a transmission electron microscope (TEM, Talos F200XS). The average diameter of the fibers was measured using a nano-measuring software. The organic composition of the composite nanofibers was measured by Fourier transform infrared spectroscopy (FTIR, Spectrum One, PerkinElmer Instruments). The hydrophilicity was evaluated using a static contact angle tester (WCA, JC-2000CD). The adsorption-desorption isotherm of BET surface area N2 was tested by a physical adsorption analyzer of ASAP2460. The confocal microscope was used to

[0091] Performance test results

[0092] 1. Morphology and properties of PRP-containing composite nanofibers

[0093] Figure 1 Figure 16 shows the SEM image of the PRP-containing composite nanofiber membrane, and the average diameter of the fibers is about 0.7 μm. Figure 2 Figure 18 is the TEM image of this kind of fiber. It can be seen from the figure that the fiber has a coaxial structure. Further staining of the fiber was carried out, and the core layer and shell layer solutions were labeled with calcein and rhodamine B, respectively (as Figure 3 shown). As 4 shown. As Figure 5As shown, under a confocal microscope, when excited at a wavelength of 495 nm, the nuclear layer can be seen as green, and when excited at a wavelength of 540 nm, the shell layer can be seen as red, confirming the core-shell fiber structure and the concentrated distribution of PRP in the core. In this study, the reason why we used the coaxial electrospinning method to prepare the structure with PRP in the core is that in traditional single-needle electrospinning, PRP will be directly mixed and contacted with organic solvents, resulting in the inactivation and denaturation of biological proteins and cytokines in PRP. By designing a core-shell structure where PRP is electrospun with an aqueous solvent inside the fiber and the outer shell layer of the fiber is electrospun with an organic solvent, the activity of PRP can be well protected. In addition, compared with single-needle electrospinning, the coaxial electrospinning method to prepare the core-shell structure can usually slow down the release rate of doped substances in the fiber, such as growth factors, drugs, etc. Slow and continuous release can usually meet the requirements of tissue repair for drug concentration.

[0094] Figure 6 is the FTIR spectrum of the composite nanofibers. In the figure, the composite nanofibers have a carboxyl stretching vibration peak of the ester group at 1725 cm -1 and a stretching vibration peak of -C-O-C- at 1245 cm -1 . These vibration peaks are from PCL. At the same time, the vibration peaks at 1640 cm -1 and 1520 cm -1 are from ε-PL. This proves that the main body of the composite nanofibers is composed of PCL and ε-PL. For coaxial fibers, in order to make the drugs in the fibers release more easily, it is hoped that the fibers have a porous structure and good wettability, so that the drugs can diffuse out in time after the tissue fluid infiltrates the fibers. Therefore, we first tested the N 2 adsorption-desorption curve of the nanofibers. As can be seen from Figure 7 , an obvious hysteresis loop appears between the adsorption and desorption curves, indicating that the nanofibers are porous structures. The fiber pore size is about 7 nm. Then, we tested the water contact angle. As can be seen from Figure 8 , the water contact angle of the PCL group is about 109°, which is a hydrophobic material. After adding ε-PL, the water contact angle changed from 109° to about 62°. The hydrophilicity of the fiber membrane was successfully changed. The fiber membrane has good hydrophilicity, can form a moist microenvironment on the wound surface, effectively absorb the exudate at the wound, and is beneficial to wound repair. Since PRP is rich in growth factors and is distributed inside the fiber core, next we evaluated the release curves of several growth factors that play a positive role in burns and scalds. Figure 9It can be seen that PDGF-BB is released faster in tissue fluid than TGF-β and VEGF. This may be because PDGF-BB tends to be water-soluble, while TGF-β and VEGF are more lipophilic. In addition, it can be seen that the content of PDGF-BB is higher than that of TGF-β and VEGF. Considering that for different individuals, the contents of these three growth factors are different, and the specific content ratio of autologous growth factors is the optimal content ratio for this person and can effectively reduce the rejection, which is also the reason why we designed to use autologous PRP.

[0095] 2. Results of the cooling experiment

[0096] The large area and deep depth of burn and scald are often due to the fact that when burn and scald occur, the local excessive heat on the skin surface cannot be quickly conducted to the air in time, resulting in heat invasion and heat diffusion to the surrounding tissues. Therefore, it is common in folk to flush with cold water at the first time of scald to relieve the local burning sensation. However, it is not easy to quickly find clean water to flush the scalded area in the wild environment, and flushing with unclean water is extremely likely to cause bacterial infection at the affected area and increase the treatment difficulty. Facing these problems, we designed to combine a portable electrospinning machine with in-situ cooling to prepare nanofibers with integrated properties of cooling, anti-inflammatory and promoting healing.

[0097] To verify the cooling effect of in-situ spun fibers, we directly deposited fibers on a heating platform simulating burn and scald at 80°C. Figure 10 The first row is the infrared thermal imaging diagram of the heating platform without any intervention (control group). It can be seen that it takes 6 minutes for the heating platform to cool from 80°C to 56°C, while directly spinning and cooling fibers on the surface of the heating platform ( Figure 10 The second row), and this kind of cooling comes from semiconductor electric refrigeration. We can see that the heating platform cools from 80°C to 35°C within 6 minutes, 21°C lower than the control group ( Figure 11 ), showing an obvious cooling effect.

[0098] As Figure 12 shown, this cooling effect may come from two mechanisms. On the one hand, the heat at the burn site is directly transferred to the cold fibers by heat conduction, using the low-temperature fibers to neutralize the excessive heat; on the other hand, the local excessive heat can also be scattered and diffused to the surrounding cooling air, and this air is colder than the surrounding temperature due to the deposition process of the cold fibers.

[0099] To verify this effect, we further scalded the back of rats and carried out in-situ spinning and cooling, Figure 13 and Figure 14 are the HE staining diagrams of the scalded parts of rats on the third day respectively. It can be seen from the figure that without cooling treatment Figure 13The number of neutrophils (appearing as dark blue cell clusters) is greater than that in in-situ electrospinning with cooling ( Figure 14 ). This indicates that by promptly reducing the temperature, local heat accumulation can be effectively reduced, which is an effective and necessary first-aid measure.

[0100] 3. In vitro antibacterial test

[0101] Burn and scald wounds are extremely vulnerable to bacterial infections. If nanofibers can kill Gram-negative bacteria and Gram-positive bacteria, it will be beneficial for the treatment of burns and scalds. Therefore, we conducted an in vitro antibacterial evaluation using the disc agar diffusion method. Figure 15 The control group in Figure 16 was untreated Escherichia coli and Staphylococcus aureus. No antibacterial zone appeared after covering with PCL fibers, indicating that PCL has no bactericidal effect on bacteria. In the other three groups, including PCL + ε-PL, PCL + PRP + ε-PL, and In-situ PCL + PRP + ε-PL, very obvious antibacterial zones appeared and the radii were basically the same, indicating that the antibacterial effect should come from ε-PL. ε-PL can interfere with the normal synthesis of the microbial cell wall, reduce the osmotic pressure resistance of the bacterial cells, and cause the bacterial cells to deform, rupture, and die. From the further SEM images of Escherichia coli and Staphylococcus aureus ( 17 ), it can be confirmed that untreated Escherichia coli has a rod-shaped structure, a smooth surface, and a plump morphology without cell damage. After treatment with PCL + PRP + ε-PL composite nanofibers, the cell membrane of the bacteria showed shrinkage, without a plump feeling, and constriction marks appeared on the cell membrane surface. Staphylococcus aureus showed similar results to Escherichia coli. This can confirm that after adding ε-PL, it can damage the cell membrane structure of microorganisms, leading to bacterial lysis and thus achieving a bactericidal effect.

[0102] 4. Biocompatibility test

[0103] Due to further in vivo applications, it is required that the PRP-containing composite nanofibers are not cytotoxic. Therefore, human fibroblasts were seeded on the nanofiber membranes to evaluate their cellular responses. Polystyrene tissue culture plates (TCP) were incubated with fibroblasts on the same day as the control group. The attachment and morphology of human fibroblasts were determined by double-label fluorescence staining of the actin cytoskeleton and the cell nucleus. Figure 18The proliferation of human skin fibroblasts (HSFs) cultured on control, PCL, PCL+ε-PL, PCL+ε-PL+PRP, and in-situ PCL+ε-PL+PRP for 1, 3, and 5 days was shown. The control group was the positive control group, and the proliferation showed an upward trend from day 1 to day 5 in each group. The PCL group showed more significant proliferation than the control group, indicating that the fibers spun from pure PCL itself had good biocompatibility. When HSFs were cultured on different samples for 1 day, the proliferation effect of HSFs on different samples was not obvious. After culturing for 3 days, the proliferation of HSFs on PCL+ε-PL was better than that on PCL. This indicated that although ε-PL was an antibacterial substance, it had no toxic effect on cells. After HSFs were cultured on different samples for 5 days, the proliferation of HSFs on PCL+ε-PL+PRP and in-situ PCL+ε-PL+PRP was significantly (P<0.01) better than that on PCL and PCL+ε-PL groups. These experimental results showed that PRP promoted the proliferation of HSFs, and these composite nanofibers were non-toxic and had no side effects on cells.

[0104] 5. Animal experiments

[0105] We evaluated the wound recovery of second-degree burns in SD rats and photographed the recovery at day 3, day 5, day 7, day 9, day 11, day 14, and day 21 respectively. Second-degree burns affect the upper and deep dermis, are white or yellow, the skin blisters and the appearance is moist. As Figure 19 shown, 3 days after the burn, the swelling of the wound surface and the redness and swelling around the wound in the control group were the most obvious, followed by the PCL group. This was because the PCL fiber membrane could prevent bacteria in the air from infecting the wound and played a protective role on the wound. 5 days after the burn, compared with the PCL group, the recovery effect of the PCL+ε-PL group was significantly better, which was attributed to the antibacterial ability of ε-PL, reducing bacterial infection and thus being beneficial to wound recovery. At day 14, obvious scabs could still be seen in the PCL+ε-PL group. In sharp contrast, the wound scabs in the PCL+ε-PL+PRP group had completely disappeared and had almost completely recovered at day 21. This indicated that the various growth factors contained in PRP were very helpful for wound healing and could effectively promote wound repair.

[0106] The fiber membranes obtained by traditional electrospinning involve two processes: spinning and using. After being cut, the fiber membranes are then covered on the wound surface. However, the fiber membranes obtained by this traditional method, similar to the use of gauze, have a poor fit with the wound. When the traditional nanofiber membrane is covered on the skin, the adhesion force between the fiber membrane and the skin is tested to be 0.02 N. In contrast, based on our handheld device using in-situ electrospinning, the fibers are directly deposited onto the skin, and the adhesion force between the fiber membrane and the skin is tested to be 0.18 N. The better fit may have an impact on the repair. Therefore, based on the PCL + ε-PL + PRP group, we further compared the differences between the traditional method and the in-situ electrospinning method for burn and scald repair. From Figure 19 , 20 , it can be seen that the in-situ group shows a faster recovery rate than the traditional electrospinning group. This may be due to the better fit, which can effectively prevent external bacteria from invading the wound through the fitting gap at the fiber membrane-skin interface, thus accelerating the burn and scald recovery. This shows that the use of the in-situ deposition method can further improve the effect of dressings containing antibacterial and growth factors. The better fit not only reduces the gap and the probability of bacterial infection, but also reduces the distance from the skin, which is conducive to the timely and effective delivery of growth factors to the wound, while enhancing the antibacterial and growth factor efficacy.

[0107] 6. Histological examination

[0108] The repair of burn and scald wounds is a very complex process. In order to analyze the relationship between wound infection and recovery from a microscopic perspective, we performed HE staining on the samples ( Figure 21) to evaluate the state of wound repair. From the 3rd to the 7th day after the burn, it can be seen that there are many lymphocytes in the control group, and relatively few in the PCL group. This shows that the PCL group has fewer lymphocytes because the coverage of the fiber membrane can protect the wound from bacterial infection. On the 7th day, the PCL+ε-PL group showed fewer lymphocytes than the PCL group. This is because ε-PL has antibacterial properties and can lyse bacteria to reduce bacteria in the wound. On the 14th day, the PCL+ε-PL+PRP group had significantly more blood vessels than the PCL+ε-PL group. This is because PRP contains a variety of growth factors. When PRP encounters tissue fluid, the Ca2+ in the tissue fluid activates PRP to release growth factors and accelerate tissue repair and regeneration. On the 21st day, the in situ PCL+ε-PL+PRP group showed more blood vessels and fewer neutrophils than the PCL+ε-PL+PRP group. This is because the fibers deposited in situ on the wound have better adhesion, which can effectively prevent external bacteria from invading the wound from the gap between the fiber membrane and the skin interface, improve antibacterial properties, and reduce the gap distance between the skin, which is conducive to the timely and effective delivery of growth factors to the wound, while improving the repair effect of growth factors on tissues. This confirms from a microscopic perspective that the in situ deposition method can further improve the effect of dressings containing antibacterial and growth factors.

[0109] We further analyzed the results of Masson's trichrome staining ( Figure 22 ) Observe the collagen deposition in the wound tissue. It can be seen that on the 3rd day, only a small amount of collagen fibers appeared in all groups. On the 7th day, the collagen fibers of all wound groups were arranged irregularly. On the 14th day, there were fewer blue collagen fibers and granulation tissue in the PCL+ε-PL group, while the collagen fibers in the PCL+ε-PL+PRP group and the in situ group PCL+ε-PL+PRP group were relatively dense. On the 21st day, the in situ PCL+ε-PL+PRP group was the first to form a complete layer of epithelial tissue on the surface of the wound tissue, indicating that tissue repair had been completed. These experimental results show that it is far from enough to rely solely on antibacterial agents to achieve the purpose of wound healing. Wound repair not only requires bactericidal effects to ablate bacteria and reduce inflammation, but also promotes wound healing through the joint action of multiple growth factors. Moreover, the in situ deposition method can further enhance the effect of dressings containing antibacterial and growth factors.

Claims

1. A preparation method of a wound dressing for burns and scalds, characterized in that, comprising: A shell solution preparation step, including dissolving polycaprolactone and polylysine in medical acetone and stirring evenly with a magnetic stirrer to obtain the shell solution; A core solution preparation step, including activating platelet-rich plasma with polyvinyl alcohol and calcium chloride to obtain the core solution; A platelet-rich plasma@polycaprolactone-polylysine core-shell nanofiber preparation step, including mixing according to the volume ratio of the shell solution to the core solution of 1:2, and using a coaxial electrospinning device for electrospinning to obtain the platelet-rich plasma@polycaprolactone-polylysine core-shell nanofiber; The coaxial electrospinning device is a handheld coaxial electrospinning device, provided with a test tube, and a semiconductor refrigerating sheet is provided on the surface of the test tube; The platelet-rich plasma@polycaprolactone-polylysine core-shell nanofiber is a core-shell fiber structure and the platelet-rich plasma is concentrated and distributed inside the core; the diameter is about 0.7 um and the pore size is about 7 nm; The N of platelet-rich plasma@polycaprolactone-polylysine core-shell nanofibers 2 There is an obvious hysteresis loop between the adsorption and desorption curves in the adsorption-desorption curve; Using the coaxial electrospinning device to electrospin the mixed solution of the shell solution and the core solution, and the obtained platelet-rich plasma@polycaprolactone-polylysine core-shell nanofiber is directly deposited on the skin.

2. The preparation method of the wound dressing for burns and scalds according to claim 1, characterized in that, The shell solution preparation step specifically includes: Dissolving 2 g of polycaprolactone and 0.4 g of polylysine in 10 g of medical acetone and stirring with a magnetic stirrer for 12 h to obtain the shell solution.

3. The preparation method of the wound dressing for burns and scalds according to claim 1, characterized in that, The core solution preparation step specifically includes: Activating platelet-rich plasma with 5% polyvinyl alcohol and calcium chloride by mass to obtain the core solution.

4. The preparation method of the wound dressing for burns and scalds according to claim 1, characterized in that, The conditions for electrospinning using the coaxial electrospinning device are: the diameter of the electrospinning nozzle is 0.4 mm, the electrospinning voltage is 10 kV, and the distance between the electrospinning nozzle and the collector is 10 cm.

5. The preparation method of the wound dressing for burns and scalds according to claim 4, characterized in that, The platelet-rich plasma@polycaprolactone-polylysine core-shell nanofiber is collected on an aluminum foil connected to the negative electrode.

6. The preparation method of the wound dressing for burns and scalds according to claim 4, characterized in that, The coaxial electrospinning device uses two alkaline batteries as the power source and is equipped with a high-voltage converter to convert 3V to 10 kV.

7. A wound dressing for burns and scalds, characterized in that, It is prepared by the preparation method of the wound dressing for burns and scalds according to any one of claims 1-6 above.

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