Wound repair material based on PRF and preparation method thereof

Abstract

The invention belongs to the technical field of wound repair, and particularly relates to a PRF-based wound repair material and a preparation method thereof. The PRF-based wound repair material is prepared from the following raw materials in parts by weight: 80 to 100 parts of PRF gel, 50 to 150 parts of Mn-MBG / quercetin composite powder and 0.5 to 2 parts of genipin, the PRF gel is used as a bioactive carrier, a natural fiber protein network is provided for building a support for cell growth and active substance release, growth factors contained in the PRF gel interact with Mn-MBG / quercetin composite powder, stable loading and slow release of quercetin are achieved, and the regulation limitation of PRF single activity on complex wounds is made up; genipin intensifies the structural stability of the PRF fiber network in a targeted manner, endows the material with a physical support capability adapted to a wound repair period, and realizes synergistic improvement of biocompatibility, antibacterial effect, healing promotion capability and structural stability.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of wound repair, and particularly relates to a wound repair material based on PRF and a preparation method thereof. BACKGROUND

[0002] Wound repair is an important topic in the medical field that has long been concerned, covering various types such as acute trauma (such as surgical incisions, burns, and abrasions), chronic refractory wounds (such as diabetic foot ulcers, pressure sores, and venous ulcers), and the like. Complications caused by wound infection and delayed healing are one of the important reasons for the increase in disability rate and mortality. The wound repair materials commonly used in current clinical practice are mainly divided into two categories: traditional non-biological materials and biological source materials, but both have significant technical defects and are difficult to meet the clinical demand for ideal wound repair materials.

[0003] PRF (platelet-rich fibrin) as the second generation of platelet concentrate has shown great potential in the field of wound repair due to its unique biological characteristics. PRF can form a fibrin scaffold with a three-dimensional network structure through optimized centrifugation process, which not only slowly releases various growth factors, but also provides a natural growth scaffold for wound repair cells and blocks bacterial invasion. However, the mechanical properties of natural PRF are poor, the morphological stability is insufficient, and the antibacterial ability is limited, further exacerbating the treatment difficulty. Therefore, the development of a composite wound repair material that can improve the mechanical properties of PRF and enhance the antibacterial activity while maintaining biocompatibility has important practical significance for solving the clinical wound healing problem and improving the treatment effect. SUMMARY

[0004] In view of the deficiencies of the prior art, the application provides a wound repair material based on PRF and a preparation method thereof. The PRF gel, Mn-MBG / quercetin composite powder, and genipin are used to prepare the material. The PRF gel serves as a biologically active carrier, providing a natural fibrin network to build a scaffold for cell growth and active substance release. The growth factors contained in the PRF gel interact with the Mn-MBG / quercetin composite powder. The mesoporous structure of the Mn-MBG composite powder realizes the stable loading and slow release of quercetin, making up for the limitations of single PRF activity in regulating complex wounds. Genipin specifically strengthens the structural stability of the PRF fibrin network, giving the material the physical support ability to adapt to the wound repair period. Ultimately, the biocompatibility, antibacterial effect, and healing ability are synergistically improved, and the structural stability is also improved. The pain points of the existing materials in complex wound repair, such as single function and poor adaptability, are comprehensively solved, and the material is particularly suitable for chronic refractory wound treatment.

[0005] The application provides a PRF-based wound repair material, which comprises the following raw materials in parts by weight: 80-100 parts of PRF gel, 50-150 parts of Mn-MBG (mesoporous bioactive glass) / quercetin composite powder, and 0.5-2 parts of genipin. The Mn-MBG / quercetin composite powder comprises the following raw materials in a mass ratio: MBG powder:MnCl2·4H2O:quercetin=1:0.4-0.6:0.05-0.1. The preparation method of the Mn-MBG / quercetin composite powder comprises the following steps: (1) MnCl2·4H2O is weighed and dissolved in deionized water to prepare a 0.1-0.5 mol / L solution, the pH value is adjusted to 5.5-6.5, MBG powder is added, constant-temperature magnetic stirring is carried out, continuous stirring is carried out at 60 DEG C for 24 h, then centrifugal separation, washing and drying are carried out to obtain a mixed powder; (2) the mixed powder is placed in a muffle furnace for calcination, heated to 600 DEG C at a temperature rising rate of 2-5 DEG C / min under an air atmosphere at room temperature, and kept at the temperature for 2 h to obtain Mn-MBG powder; (3) quercetin is weighed and dissolved in anhydrous ethanol to prepare a stock solution with a concentration of 1.0-2.0 mg / mL, the operation is carried out in the dark, Mn-MBG powder is added to obtain a suspension, the suspension is placed in a shaking bed at room temperature and in the dark for low-speed oscillation for 24-48 h to obtain a mixed product; (4) the mixed product is centrifuged, the solid product is collected, washing is carried out to remove the quercetin physically adsorbed on the surface, the washed product is transferred to a culture dish and placed in a light-proof vacuum dryer, dried at room temperature or 30 DEG C for 24 h until completely dried to obtain Mn-MBG / quercetin composite powder.

[0006] The preparation method of the PRF gel comprises the following steps: (a) a venous blood sample is obtained and immediately injected into a vacuum blood collection tube without any anticoagulant, the vacuum blood collection tube is placed in a centrifuge, and an optimized gradient centrifugation method is used to obtain a centrifugal product; (b) the PRF clot in the form of middle amber color and gel is carefully taken out from the centrifugal product with a sterile forceps, washed with normal saline, and the red blood cell layer adhered to the bottom is carefully removed, the pure PRF clot is transferred to a sterile beaker, and a homogenizer is used for homogenization treatment to obtain a uniform and viscous PRF gel.

[0007] The application further provides a preparation method of the PRF-based wound repair material, which specifically comprises the following steps: S1, Mn-MBG / quercetin composite powder was weighed and added into PRF gel, ice PBS buffer was added, and the mixture was stirred magnetically in the dark for 45 min to form a uniform viscous composite slurry; S2, the composite slurry was injected into a sterile mold, the surface was scraped flat with a sterile spatula, the mold was gently shaken to remove large bubbles, the sterile mold was placed in a 4℃ refrigerator for 1 h for preliminary shaping, and then taken out and quickly transferred to a-80℃ ultra-low temperature refrigerator or liquid nitrogen for rapid freezing for 6-12 h until complete freezing to form a frozen sample; S3, the frozen sample was quickly moved into the sample compartment of a freeze dryer, and freeze-dried for 36-48 h until all the water in the sample completely sublimated to obtain a porous sponge body; S4, genipin was weighed and dissolved in PBS buffer with pH 7.4, and stirred in the dark until completely dissolved to form a genipin crosslinking solution with a mass-volume concentration of 0.2-0.3%, the porous sponge body was soaked in the genipin crosslinking solution to ensure complete immersion, and crosslinked at 37℃ in the dark with gentle shaking for 12 h, after crosslinking, the sample was washed with ultrapure water to completely remove unreacted genipin, and then freeze-dried again for 24 h at a temperature of-55℃ and a vacuum degree of <0.1 mbar to obtain a PRF-based wound repair material.

[0008] Compared with the prior art, the present application has the following beneficial effects: The PRF gel is used as a bioactive core carrier, which can not only release key growth factors to promote wound healing and provide basic conditions for cell proliferation and angiogenesis, but also become an ideal dispersion carrier for the Mn-MBG / quercetin composite powder due to its viscous properties, avoiding uneven function caused by the aggregation of the composite powder; the Mn-MBG / quercetin composite powder is used as a functional component, the mesoporous structure of Mn-MBG can realize the stable loading and release of active substances, which not only makes up for the insufficient regulation ability of PRF single activity on complex wounds, but also prolongs the action period of active substances through the complementation of the mesoporous network and the fiber structure of the PRF gel. Genipin is used as a mild crosslinking agent to only strengthen the structural stability of PRF fibrin, without damaging the activity of PRF growth factors, Mn-MBG ions and quercetin, so that the material has physical support ability suitable for the wound repair period while maintaining biocompatibility, further strengthening the effects of promoting blood vessels and anti-inflammatory. In the wound healing process, the drug effects of PRF and Mn-MBG / quercetin composite powder are mutually synergistic, which can not only effectively regulate the inflammatory response of the wound, break the healing stagnation state caused by long-term inflammation of chronic wounds, but also significantly promote angiogenesis, improve the ischemic and hypoxic environment of the wound, and inhibit the growth of pathogenic bacteria, reducing the risk of infection; the porous structure and suitable mechanical properties of the material can provide sustained physical protection for the wound, which is not easy to break and can avoid the interruption of healing caused by material shedding or structural failure; the long-acting release characteristics of active substances can cover the long healing period of chronic wounds, avoiding the obstruction of the healing process caused by active supply interruption. The whole preparation process cooperates with each other to provide protection for raw material synergy; maintain long-acting release of active substances, effectively cope with the complex pathological environment of chronic wounds, significantly improve the clinical repair effect, and provide a more comprehensive and efficient solution for chronic wound treatment. BRIEF DESCRIPTION OF DRAWINGS

[0009] Figure 1 H&E staining of skin tissue at day 14 of the wound repair material prepared in Example 2; Figure 2 H&E staining of skin tissue at 7 days and 14 days of the control group and the wound repair material prepared in Example 2; Figure 3 Inflammation factor expression chart of the control group and the wound repair material prepared in Example 2. DETAILED DESCRIPTION

[0010] In order to make the person skilled in the art better understand the technical solutions of the present application, and make the above-mentioned features, objects and advantages of the present application more clear and easy to understand, the present application will be further described below in combination with examples. The examples are only used to illustrate the present application and not to limit the scope of the present application.

[0011] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Also, any method and material similar or equivalent to those described herein can be used in the practice of the present application. The described preferred methods and materials are only given by way of example and are not intended to limit the content of the present application.

[0012] In the following examples, unless otherwise specified, the methods are conventional methods; in the following examples, unless otherwise specified, the materials used are commercially available new materials.

[0013] Example 1: The present example provides a wound repair material based on PRF, which comprises the following raw materials in parts by weight: 80 parts of PRF gel, 50 parts of Mn-MBG / quercetin composite powder, 0.5 parts of genipin; The Mn-MBG / quercetin composite powder comprises the following raw materials in mass ratio: MBG powder: MnCl2·4H2O: quercetin = 1:0.4:0.05; The preparation method of the Mn-MBG / quercetin composite powder comprises the following steps: (1) Weigh MnCl2·4H2O, dissolve it in deionized water to prepare a 0.1 mol / L solution, adjust the pH to 5.5 with acetic acid, add MBG powder, constant temperature magnetic stirring, continuous stirring at 300 rpm for 24 h at 60 ℃, then centrifuge at 10000 rpm for 10 min, wash with ultrapure water and anhydrous ethanol alternately for 3 times, vacuum dry at 60 ℃ for 12 h, to obtain a mixed powder; (2) Place the mixed powder in a muffle furnace for calcination, heat to 600 ℃ at a heating rate of 2 ℃ / min under air atmosphere at room temperature, and keep the temperature for 2 h to obtain Mn-MBG powder; (3) Weigh quercetin, dissolve it in anhydrous ethanol to prepare a stock solution with a concentration of 1.0 mg / mL, operate in the dark, add Mn-MBG powder to obtain a suspension, place the suspension in a light-proof container, seal with sealing film, and place it in a shaking bed at a low speed of 100 rpm for 24 h at room temperature and in the dark to obtain a mixed product; (4) Centrifuge the mixed product at 10000 rpm for 10 min, collect the solid product, quickly rinse the precipitate once with a small amount of ice-cold anhydrous ethanol to remove the physically adsorbed quercetin on the surface, transfer the washed product to a culture dish, place it in a vacuum dryer, avoid light, dry at room temperature for 24 h until completely dry, to obtain the final Mn-MBG / quercetin composite powder.

[0014] The preparation method of the PRF gel comprises the following steps: (a) Obtain a venous blood sample, immediately inject it into a vacuum blood collection tube without any anticoagulant, place the vacuum blood collection tube in a centrifuge, and use an optimized gradient centrifugation method to centrifuge at 700 g for 3 min and then at 300 g for 8 min at room temperature to obtain a centrifugation product; (b) Carefully take out the PRF clot in the middle of the centrifugation product with sterile tweezers, put it into a sterile culture dish, gently rinse it with normal saline, and carefully remove the red blood cell layer adhered to the bottom, transfer the pure PRF clot to a sterile beaker, and use a homogenizer to process it into a uniform and viscous PRF gel under ice bath conditions at a speed of 1000 rpm for 10 s each time for 3 times.

[0015] The embodiment also provides a preparation method of a PRF-based wound repair material, which specifically comprises the following steps: S1, weigh the Mn-MBG / quercetin composite powder and add it into the PRF gel, add ice PBS buffer, and magnetically stir at a speed of 500 rpm under ice bath conditions for 45 min in the dark to form a uniform and viscous composite slurry; S2, inject the composite slurry into a sterile mold, flatten the surface with a sterile spatula, gently shake the mold to remove large bubbles, and place the sterile mold in a 4 ℃ refrigerator for 1 h to allow the fibrinogen of the PRF to further polymerize and preliminarily shape, take out, and quickly transfer to a-80 ℃ ultra-low temperature refrigerator for rapid freezing for 6 h until complete freezing to form a frozen sample; S3, quickly move the frozen sample into the sample compartment of a freeze dryer, and freeze dry at-55 ℃ and a vacuum degree of 0.05 mbar for 36 h until all the water in the sample is completely sublimated to obtain a primary porous sponge; S4, dissolve genipin in a PBS buffer with a pH of 7.4, stir in the dark until completely dissolved to form a genipin crosslinking solution with a mass-volume concentration of 0.2%, soak the porous sponge in the genipin crosslinking solution to ensure complete immersion, and crosslink at 37 ℃ in the dark under gentle oscillation for 12 h, after the crosslinking is completed, wash with ultrapure water to completely remove the unreacted genipin, and freeze dry again for 24 h at a temperature of-55 ℃ and a vacuum degree of 0.05 mbar to obtain the PRF-based wound repair material.

[0016] Embodiment 2: The embodiment provides a PRF-based wound repair material, which comprises the following raw materials in parts by weight: 90 parts of PRF gel, 100 parts of Mn-MBG / quercetin composite powder, and 1.2 parts of genipin. The Mn-MBG / quercetin composite powder comprises raw materials in the following mass ratio: MBG powder: MnCl2·4H2O: quercetin = 1:0.5:0.08; The preparation method of the Mn-MBG / quercetin composite powder comprises the following steps: (1) MnCl2·4H2O is weighed and dissolved in deionized water to prepare a 0.2 mol / L solution, and acetic acid is used to adjust the pH to 5.5. MBG powder is added, constant temperature magnetic stirring is carried out, and continuous stirring is carried out at 300 rpm for 24 h at 60°C. Then, 10000 rpm centrifugal speed is used for centrifugation for 10 min, and the mixed powder is washed with ultrapure water and anhydrous ethanol for 3 times, and vacuum drying is carried out at 60°C for 12 h to obtain the mixed powder; (2) The mixed powder is placed in a muffle furnace for calcination, heated to 600°C at a heating rate of 3°C / min under air atmosphere at room temperature, and kept at this temperature for 2 h to obtain Mn-MBG powder; (3) Quercetin is weighed and dissolved in anhydrous ethanol to prepare a stock solution with a concentration of 1.5 mg / mL, and the operation is carried out in the dark. Mn-MBG powder is added to obtain a suspension, and the suspension is placed in a light-proof container and sealed with sealing film. The suspension is placed in a shaking bed at room temperature and in the dark at a low speed of 100 rpm for 36 h to obtain a mixed product; (4) The mixed product is centrifuged at a speed of 10000 rpm for 10 min, and the solid product is collected. The precipitate is quickly rinsed with a small amount of ice-cold anhydrous ethanol for 2 times to remove the quercetin physically adsorbed on the surface. The washed product is transferred to a culture dish and placed in a vacuum dryer in the dark at room temperature for 24 h until completely dried to obtain the final Mn-MBG / quercetin composite powder.

[0017] The preparation method of the PRF gel comprises the following steps: (a) A venous blood sample is obtained and immediately injected into a vacuum blood collection tube without any anticoagulant. The vacuum blood collection tube is placed in a centrifuge, and an optimized gradient centrifugation method is used. The centrifuge is first centrifuged at 700 g for 3 min, and then centrifuged at 300 g for 8 min at room temperature to obtain a centrifuged product; (b) The centrifuged product is carefully taken out with sterile tweezers to obtain a middle amber PRF gel block, which is placed in a sterile culture dish and washed with normal saline. The bottom adhered red blood cell layer is carefully removed, and the pure PRF gel block is transferred to a sterile beaker. A homogenizer is used to process the PRF gel block into a uniform and viscous PRF gel under ice bath conditions at a speed of 1000 rpm for 10 s each time, repeated 4 times.

[0018] The embodiment also provides a preparation method of the wound repair material based on PRF, and specifically comprises the following steps: S1, Mn-MBG / quercetin composite powder is weighed and added into PRF gel, ice PBS buffer is added, and ice bath conditions are used for magnetic stirring at a speed of 500 rpm in the dark for 45 min to form a uniform viscous composite slurry; S2, the composite slurry is injected into a sterile mold, the surface is flattened with a sterile spatula, the mold is gently shaken to remove large bubbles, and the sterile mold is placed in a 4 ℃ refrigerator for 1 h to allow the fibrinogen of PRF to further polymerize, preliminary shaping is performed, the mold is taken out and quickly transferred to a-80 ℃ ultra-low temperature refrigerator, and quick freezing is performed for 8 h until complete freezing to form a frozen sample; S3, the frozen sample is quickly moved into a sample bin of a freeze dryer, and freeze drying is performed at-55 ℃ and a vacuum degree of 0.05 mbar for 36 h until all the water in the sample is completely sublimated to obtain a primary porous sponge body; S4, genipin is weighed and dissolved in a PBS buffer with a pH of 7.4, stirred in the dark until completely dissolved to form a genipin crosslinking solution with a mass-volume concentration of 0.25%, the porous sponge body is soaked in the genipin crosslinking solution to ensure complete immersion, and crosslinking is performed at 37 ℃ in the dark under gentle oscillation for 12 h; after the crosslinking is completed, the unreacted genipin is completely removed by washing with ultrapure water, and freeze drying is performed again for 24 h at a temperature of-55 ℃ and a vacuum degree of 0.05 mbar to obtain the wound repair material based on PRF.

[0019] Embodiment 3: The embodiment provides a wound repair material based on PRF, and the wound repair material comprises the following raw materials in parts by weight: 100 parts of PRF gel, 150 parts of Mn-MBG / quercetin composite powder, and 2 parts of genipin. The Mn-MBG / quercetin composite powder comprises the following raw materials in a mass ratio: MBG powder:MnCl2·4H2O:quercetin=1:0.6:0.1; The preparation method of the Mn-MBG / quercetin composite powder comprises the following steps: (1) MnCl2·4H2O is weighed and dissolved in deionized water to prepare a 0.5 mol / L solution, acetic acid is used to adjust the pH to 6.5, MBG powder is added, constant-temperature magnetic stirring is performed, continuous stirring is performed at a speed of 300 rpm at 60 ℃ for 24 h, then centrifugation is performed at a speed of 10,000 rpm for 10 min, the mixed powder is washed with ultrapure water and anhydrous ethanol alternately for 3 times, and vacuum drying is performed at 60 ℃ for 12 h to obtain the mixed powder; (2) Put the mixed powder into a muffle furnace for calcination, heat to 600 ℃ at a temperature rising rate of 5 ℃ / min under air atmosphere at room temperature, and keep the temperature for 2 h to obtain Mn-MBG powder; (3) Weigh quercetin, dissolve it in anhydrous ethanol to prepare a stock solution with a concentration of 2.0 mg / mL, operate in the dark, add Mn-MBG powder to obtain a suspension, and place the suspension in a light-proof container, seal it with sealing film, and place it in a shaking bed at room temperature and in the dark at a low speed of 100 rpm for 48 h to ensure that the quercetin molecules are fully diffused and adsorbed into the mesoporous channels to obtain a mixed product; (4) Centrifuge the mixed product at a speed of 10,000 rpm for 10 min, collect the solid product, quickly rinse the precipitate with a small amount of ice-cold anhydrous ethanol twice to remove the physically adsorbed quercetin on the surface, transfer the washed product to a culture dish, and dry it in a vacuum dryer at room temperature for 24 h in the dark until completely dry to obtain the final Mn-MBG / quercetin composite powder.

[0020] The preparation method of the PRF gel comprises the following steps: (a) Obtain a venous blood sample, immediately inject it into a vacuum blood collection tube containing no anticoagulant, place the vacuum blood collection tube in a centrifuge, and use an optimized gradient centrifugation method to first centrifuge at 700 g for 3 min and then centrifuge at 300 g for 8 min at room temperature to obtain a centrifuged product; (b) Carefully take out the PRF clot in the middle of the centrifuged product with sterile tweezers, put it into a sterile culture dish, rinse it gently with normal saline, and carefully remove the red blood cell layer adhered to the bottom, transfer the pure PRF clot to a sterile beaker, and use a homogenizer to process it into a uniform and viscous PRF gel under ice bath conditions at a speed of 1000 rpm for 10 s each time, repeated 4 times.

[0021] The embodiment also provides a preparation method of a PRF-based wound repair material, which specifically comprises the following steps: S1, weigh the Mn-MBG / quercetin composite powder and add it to the PRF gel, add ice-cold PBS buffer, and magnetically stir at a speed of 500 rpm in the dark for 45 min to form a uniform and viscous composite slurry; S2, inject the composite slurry into a sterile mold, flatten the surface with a sterile spatula, gently shake the mold to remove large bubbles, place the sterile mold in a 4 ℃ refrigerator for 1 h to allow the fibrinogen of PRF to further polymerize and be initially shaped, take it out, quickly transfer it to a-80 ℃ ultra-low temperature refrigerator, and freeze it quickly for 12 h until it is completely frozen to form a frozen sample. S3, the frozen sample is quickly moved into the sample chamber of the freeze dryer, and freeze-dried at -55 ℃ and a vacuum degree of 0.08 mbar for 48 h until all the water in the sample is completely sublimated, to obtain a primary porous sponge body; S4, genipin is weighed and dissolved in PBS buffer with a pH of 7.4, stirred in the dark until completely dissolved, forming a genipin cross-linking solution with a mass-volume concentration of 0.3%, the porous sponge body is soaked in the genipin cross-linking solution, ensuring complete immersion, and cross-linked at 37 ℃ in the dark with gentle shaking for 12 h. After cross-linking is completed, the unreacted genipin is completely removed by washing with ultrapure water, and freeze-dried again for 24 h at a temperature of -55 ℃ and a vacuum degree of 0.08 mbar, to obtain a wound repair material based on PRF.

[0022] Comparative Example 1 differs from Example 2 in that no Mn-MBG / quercetin composite powder is added, and the rest is exactly the same as Example 2.

[0023] Comparative Example 2 differs from Example 2 in that no MBG is added, and the rest is exactly the same as Example 2.

[0024] Comparative Example 3 differs from Example 2 in that no quercetin is added, and the rest is exactly the same as Example 2.

[0025] Experimental Example: 1. Cytotoxicity test: MTT method is used for cytotoxicity test, and the wound repair materials prepared by Examples 1-3 and Comparative Examples 1-3 are used as samples. The samples are cut into small pieces of 10 mm x 10 mm x 2 mm, washed gently with sterile normal saline for 3 times to remove surface residues; in a hundred-level sterile operation table, the samples are placed in sterile centrifuge tubes, 10% fetal bovine serum (FBS) containing DMEM medium is added, sealed and placed in a 37 ℃, 5% CO2 incubator for static extraction for 24 h to obtain the extraction solution; after extraction is completed, the extraction solution is filtered with a 0.22 μm sterile filter membrane to remove possible microorganisms or particles for standby use (at the same time, blank extraction medium without samples is prepared as negative control, and 0.1% Triton X-100 containing medium is prepared as positive control). Prepare human skin fibroblasts (HSF) and umbilical vein endothelial cells (HUVEC) with 10% FBS, 1% penicillin-streptomycin-containing DMEM medium, and culture in a 37 ℃, 5% CO2 incubator until the logarithmic growth phase; the cells are digested with 0.25% trypsin, and the cell concentration is adjusted to 5×10 4The MTT solution was added to each well of the 96-well plate at 100 μL, and incubation was continued for 4 h. The liquid in the wells was discarded, and dimethyl sulfoxide was added to dissolve the formazan crystals. The absorbance value (OD value) was measured at a wavelength of 570 nm using an enzyme-labeled instrument. The cell survival rate was calculated, and the cell survival rate (%) = (A test group - A blank group) / (A negative control group - A blank group) x 100%. The results are shown in Table 1.

[0026] 2. Mechanical property test: The wound repair materials prepared in Examples 1-3 and Comparative Examples 1-3 were used as test samples, and the compressive strength was tested. The test sample was cut into a standard piece with a size of 10 mm x 10 mm. A universal material testing machine was used, and the sample was placed in the center of the lower press plate of the testing machine to ensure that the upper and lower press plates were perpendicular to the sample. The test was started, and the machine automatically applied pressure until the sample was crushed, and the test was stopped. The maximum pressure value was recorded, and the compressive strength was calculated according to the formula: compressive strength = maximum pressure / sample pressure area. The average of three values was taken, and the results are shown in Table 1. The tensile test mode was switched, and the loading rate was set to 1 mm / min. The dumbbell-shaped sample was clamped at both ends of the testing machine, and the sample axis was ensured to be consistent with the tensile direction. The test was started, and the machine stretched the sample to break, and the tensile force-displacement curve was automatically recorded. The maximum tensile force at break was read from the curve, and the tensile strength = maximum tensile force / sample parallel section area was calculated. The data of the elastic stage (initial linear segment) of the curve were taken, and the elastic modulus = stress change / strain change was calculated. The average of three values was taken, and the results are shown in Table 1.

[0027] Table 1: Cell survival rate and mechanical property test results

[0028] As shown in Table 1, the cell survival rates of Examples 1-3 were all above 98.5%, while the cell survival rates of Comparative Examples 1-2 were only 90.5%-92.1%, and the cell survival rate of Comparative Example 3 was 94.0%, indicating that the examples had better biocompatibility and less negative impact on cell growth and survival, and were more conducive to cell adhesion and proliferation on the material. The compressive strength of Examples 1-3 was about 1.15-1.20 MPa, the tensile strength was about 2.16-2.25 MPa, and the elastic modulus was about 8.85-9.21 MPa; the compressive strength of Comparative Examples 1-2 was only 0.25-0.26 MPa, the tensile strength was 0.61-0.68 MPa, and the elastic modulus was 1.98-2.15 MPa, which was much lower than that of the examples; although the mechanical properties of Comparative Example 3 were relatively close to those of the examples, the cell survival rate was still lower than that of the examples, indicating that the wound repair material prepared in the present application could provide better structural support for application scenarios such as wound repair.

[0029] 3. Antibacterial performance: The wound repair materials prepared from Examples 1-3 and Comparative Examples 1-3 were used as samples, and the plate counting method was used to test the antibacterial rate. Staphylococcus aureus and Escherichia coli, which are common pathogenic bacteria of wounds, were tested. The pathogenic bacteria were inoculated into LB liquid medium, and cultured at 37°C with 180 rpm shaking until the logarithmic growth phase (the concentration of the bacterial solution was adjusted to 1x10 6 CFU / mL). Sterilized samples (cut into 1 cm x 1 cm pieces) were added to the bacterial solution at a ratio of 1 g:10 mL. A blank control group (only bacterial solution) and a sterile dressing control group (medical gauze + bacterial solution) were set up, with 3 parallel samples in each group. The samples were incubated at 37°C for 24 hours. After incubation, 1 mL of bacterial solution was diluted by a factor of 10 -5 in each group, and 100 μL of the diluted solution was evenly spread on LB solid medium plates. The plates were incubated at 37°C for 18 hours, and then the number of colonies (CFU) was counted. The antibacterial rate was calculated using the formula: antibacterial rate (%) = (average number of colonies in the control group - average number of colonies in the test group) / average number of colonies in the control group x 100%. The results are shown in Table 2.

[0030] 4. Wound healing rate: The wound repair materials prepared from Examples 1-3 and Comparative Examples 1-3 were used as samples. Healthy SD rats were selected, and streptozotocin (STZ) was injected intraperitoneally to establish a diabetic model (blood glucose ≥ 16.7 mmol / L was considered successful modeling). After 1 week of stable feeding, the rats were randomly divided into a test group, a blank control group (without material), and a normal dressing control group (with medical gauze). Each group had at least 6 rats. After intraperitoneal injection of anesthetic, the rats' back hair was shaved and sterilized. A sterile surgical ring (1 cm in diameter) was used to locate, incise, and remove full-thickness skin (deep to the fascia) to form a standard wound (initial area of about 0.785 cm 2 ). The test group was applied with sterilized sample material. Each group was covered with gauze and fixed with tape, and then single-caged. On the 3rd, 7th, 14th, and 21st days after modeling, the rats were lightly anesthetized, the dressing was removed, and a digital camera with a transparent ruler was used to take photos of the wound under uniform conditions. After taking the photos, the corresponding materials / dressings were replaced and sterilized. The remaining wound area was measured using ImageJ software calibrated with the ruler. The average healing rate of each group was calculated using the formula: wound healing rate (%) = (initial wound area - remaining wound area) / initial wound area x 100%. The results are shown in Table 2.

[0031] Table 2: Results of antibacterial properties and wound healing rates

[0032] As shown in the results of Table 2, the bacteriostatic rates of Examples 1-3 against S. aureus and E. coli are generally 98% and above, while the bacteriostatic rates of Comparative Examples 1-2 are significantly lower, only about 45%-55%, and the bacteriostatic rate of Comparative Example 3 is also relatively high, but still lags behind Examples. It is shown that the wound repair material prepared in the present application can effectively inhibit pathogenic bacteria such as S. aureus and E. coli, and has excellent anti-infection effect; the wound healing rates of Examples 1-3 are all 98.9% and above, and the healing rates of Comparative Examples 1-3 are far lower than those of Examples 1-3. It is shown that the wound repair material prepared in the present application can better promote wound healing, and has obvious advantages in wound repair effect.

[0033] Figure 1 It is shown that the PRF-based wound repair material prepared in Example 2 has continuous and moderately thick new epithelium and more mature fibers at 14 days; Figure 2 The skin tissue H&E staining of the control group and the PRF-based wound repair material prepared in Example 2 at 7 days and 14 days is shown in the figure. It can be seen that at 7 days, Example 2 has more granulation tissue and epithelialization than the control group, has less inflammatory cell infiltration of skin tissue, and has more ordered tissue arrangement; at 14 days, the epidermal layer of Example 2 is more complete, has more uniform thickness, has continuous and moderately thick new epithelium at the center and edge of the skin tissue, while the repair degree of the skin tissue of the control group is relatively lagging behind, and the epidermal integrity is poor. It can be seen that the PRF-based wound repair material prepared in Example 2 can more effectively promote the repair and regeneration of skin tissue and accelerate the wound healing process at 7 days and 14 days. Figure 3 The inflammation factor expression chart of the control group and the PRF-based wound repair material prepared in Example 2 is shown. At 7 days, it can be known that the expression of the anti-salt gene TGF-β in Example 2 is increased, and the expressions of the inflammation genes IL-6 and TNF-α are reduced, which shows that the PRF-based wound repair material prepared in the present application promotes the healing of tissue trauma and plays an anti-inflammatory role.

[0034] In summary, the PRF-based wound repair material prepared in the present application exhibits excellent and comprehensive performance in terms of biocompatibility, mechanical properties, bacteriostatic effect and wound healing promotion; it not only retains the growth factor activity of PRF, the anti-inflammatory and antibacterial effects of Mn-MBG / quercetin and the proangiogenic effect, but also optimizes the material structure through the step of genipin crosslinking, thereby providing multi-dimensional effective support for wound repair; in terms of function, it can efficiently inhibit pathogenic bacteria and significantly promote wound healing. Each link in the preparation process is closely coordinated from the retention of raw material activity to the optimization of material structure, thereby providing an effective and comprehensive solution for wound repair, especially for the treatment of chronic refractory wounds, and having good application prospect.

[0035] The above merely describes preferred specific embodiments of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art, according to the technical solution and inventive concept of the present application, makes equivalent replacement or change within the technical range disclosed by the present application, which should be covered within the protection scope of the present application.

Claims

1. A PRF-based wound repair material, characterized in that, The ingredients include the following parts by weight: 80-100 parts PRF gel, 50-150 parts Mn-MBG / quercetin complex powder, and 0.5-2 parts genipin; The Mn-MBG / quercetin composite powder comprises raw materials in the following mass ratio: MBG powder:MnCl2·4H2O:quercetin = 1:0.4-0.6:0.05-0.1; The preparation method of the Mn-MBG / quercetin composite powder includes the following steps: (1) Weigh MnCl2·4H2O, dissolve it in deionized water to form a solution, adjust the pH, add MBG powder, stir magnetically at constant temperature, then centrifuge, wash, dry and obtain mixed powder; (2) The mixed powder was calcined to obtain Mn-MBG powder; (3) Weigh quercetin, dissolve it in anhydrous ethanol, protect it from light, prepare a stock solution, add Mn-MBG powder to obtain a suspension, and shake the suspension at low speed at room temperature and in the dark to obtain a mixed product. (4) The mixed product was centrifuged, washed, and dried to obtain Mn-MBG / quercetin composite powder.

2. The PRF-based wound repair material according to claim 1, characterized in that, In step (1), the concentration of the solution is 0.1-0.5 mol / L; in step (3), the concentration of the stock solution is 1.0-2.0 mg / mL.

3. The PRF-based wound repair material according to claim 1, characterized in that, The method for preparing the PRF gel includes the following steps: (a) Obtain a venous blood sample, centrifuge it, and obtain the centrifuged product; (b) Remove the PRF clump from the centrifuged product, rinse the PRF clump, and homogenize it to obtain PRF gel.

4. A method for preparing a PRF-based wound repair material according to any one of claims 1-3, characterized in that, Specifically, the following steps are included: S1, Weigh out Mn-MBG / quercetin composite powder and add it to PRF gel, add ice-cold PBS buffer, stir in the dark to form composite slurry; S2, the composite slurry is injected into the mold, allowed to stand at low temperature for preliminary shaping, and then rapidly frozen to form a frozen sample; S3. Freeze-dry the frozen sample to obtain a primary porous sponge. S4, weigh genipin and prepare genipin crosslinking solution. Crosslink the porous sponge with genipin crosslinking solution in the dark. After washing, freeze-dry again to obtain PRF-based wound repair material.

5. The method for preparing a PRF-based wound repair material according to claim 4, characterized in that, In step S4, the preparation process of the genipin crosslinking solution is as follows: dissolve genipin in PBS buffer with a pH of 7.4, stir in the dark, and obtain a genipin crosslinking solution with a mass-volume concentration of 0.2-0.3%.

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