A prf-based wound repair material and a preparation method thereof

Abstract

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; wherein the wound repair material based on PRF comprises the following raw materials in parts by weight: 80-100 parts of PRF gel, 50-150 parts of Mn-MBG / quercetin composite powder, and 0.5-2 parts of genipin; the PRF gel serves as a bioactive carrier, which not only provides a natural fibrin network to build a scaffold for cell growth and active substance release, but also realizes stable loading and slow release of quercetin by interaction between growth factors contained in the PRF gel and the Mn-MBG / quercetin composite powder, so as to make up for the regulation limitation of single activity of PRF on complex wounds; the genipin specifically strengthens the structural stability of the PRF fibrin network, endows the material with physical support ability suitable for the wound repair period, and realizes synergistic improvement of biocompatibility, antibacterial effect, healing promotion ability 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 for a long time, and covers 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), etc. The current clinically commonly used wound repair materials are mainly divided into two categories of traditional non-biological materials and biological source materials, but both have significant technical defects, and it is difficult to meet the clinical demand for ideal wound repair materials.

[0003] PRF (platelet-rich fibrin) as the second generation of platelet concentrate has 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 optimization of the centrifugal process, and the scaffold can not only slowly release various growth factors, but also provide a natural growth scaffold for wound repair cells and block bacterial invasion. However, the natural PRF has poor mechanical properties, insufficient morphological stability, and limited antibacterial ability, which further exacerbates the treatment difficulty. Therefore, it is of great practical significance to develop a composite wound repair material that can improve the mechanical properties of PRF and enhance the antibacterial activity while maintaining biocompatibility, in order to solve the clinical wound healing problem and improve 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 wound repair material based on PRF. The PRF gel serves as a bioactive carrier, which not only provides a natural fibrin network to build a scaffold for cell growth and release of active substances, but also interacts 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 activity of PRF in regulating complex wounds. Genipin specifically strengthens the structural stability of the PRF fibrin network and endows the material with physical support ability adapted to the wound repair period. Finally, the biocompatibility, antibacterial effect, healing ability and structural stability are synergistically improved, and the pain points of the existing materials in complex wound repair, such as single function and poor adaptability, are solved comprehensively, and the material is especially suitable for chronic refractory wound treatment.

[0005] The application provides a wound repair material based on PRF, 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.

[0006] 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;

[0007] The preparation method of the Mn-MBG / quercetin composite powder comprises the following steps:

[0008] (1) MnCl2·4H2O is weighed and dissolved in deionized water to prepare a solution with a concentration of 0.1-0.5 mol / L, the pH is adjusted to 5.5-6.5, and then MBG powder is added, constant-temperature magnetic stirring is carried out, continuous stirring is carried out at 60℃ for 24 h, and then centrifugation, washing and drying are carried out to obtain a mixed powder;

[0009] (2) The mixed powder is placed in a muffle furnace for calcination, heated to 600℃ at a temperature increasing rate of 2-5 ℃ / min under an air atmosphere at room temperature, and kept at the temperature for 2 h to obtain Mn-MBG powder;

[0010] (3) Quercetin is weighed and dissolved in anhydrous ethanol to prepare a stock solution with a concentration of 1.0-2.0 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 shaking bed at room temperature and in the dark for low-speed oscillation for 24-48 h to obtain a mixed product;

[0011] (4) The mixed product is centrifuged, the solid product is collected, and washing is carried out to remove quercetin physically adsorbed on the surface, the washed product is transferred to a culture dish and placed in a light-proof vacuum dryer, and dried at room temperature or 30℃ for 24 h until completely dried to obtain Mn-MBG / quercetin composite powder.

[0012] The preparation method of the PRF gel comprises the following steps:

[0013] (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 centrifuged product;

[0014] (b) The PRF clot in the form of an intermediate amber color and a gel is carefully taken out from the centrifuged product with sterile tweezers, placed in a sterile culture dish, washed with normal saline, and the red blood cell layer adhered to the bottom is carefully removed, and the pure PRF clot is transferred to a sterile beaker and homogenized to obtain a uniform and viscous PRF gel.

[0015] The application also provides a preparation method of a PRF-based wound repair material, which specifically comprises the following steps:

[0016] S1, Mn-MBG / quercetin composite powder is weighed and added into PRF gel, ice PBS buffer is added, and the mixture is stirred magnetically in the dark for 45 min to form a uniform viscous composite slurry;

[0017] S2, the composite slurry is injected into a sterile mold, the surface is scraped flat with a sterile spatula, the mold is gently shaken to remove large bubbles, the sterile mold is placed in a 4 DEG C refrigerator for 1 h for preliminary shaping, and then taken out and quickly transferred to a-80 DEG C ultra-low temperature refrigerator or liquid nitrogen for rapid freezing for 6-12 h until complete freezing to form a frozen sample;

[0018] S3, the frozen sample is quickly moved into a sample bin of a freeze dryer, and freeze-dried for 36-48 h until all water in the sample is completely sublimated to obtain a porous sponge body;

[0019] 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 0.2-0.3%, the porous sponge body is soaked in the genipin crosslinking solution to ensure complete immersion, and crosslinked at 37 DEG C in the dark under gentle shaking for 12 h, after crosslinking, the sample is washed with ultrapure water to completely remove unreacted genipin, and then freeze-dried again for 24 h at a temperature of-55 DEG C and a vacuum degree of <0.1 mbar to obtain a PRF-based wound repair material.

[0020] Compared with the prior art, the application has the beneficial effects as follows:

[0021] 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, and 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 complements the fiber structure of PRF gel through the mesoporous network, prolonging the action period of active substances. 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 pro-vascular and anti-inflammatory effects. 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 longer 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 cooperation; 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

[0022] Figure 1 Skin tissue H&E staining of the wound repair material prepared in Example 2 at day 14;

[0023] Figure 2 Skin tissue H&E staining of the wound repair material prepared in Example 2 and the control group at 7 days and 14 days;

[0024] Figure 3 Inflammation factor expression chart of the wound repair material prepared in Example 2 and the control group. DETAILED DESCRIPTION

[0025] In order to make the technical scheme of the present application better understood by the person skilled in the art, 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 conjunction with the embodiments. The embodiments are only used to illustrate the present application and not to limit the scope of the present application.

[0026] 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. In addition, any method and material similar or equivalent to those described herein can be used in the present application. The preferred methods and materials described herein are only exemplary and not intended to limit the scope of the present application.

[0027] In the following examples, unless otherwise specified, all are conventional methods; the materials used in the following examples, unless otherwise specified, the raw materials are market-purchased new materials.

[0028] Example 1: The present embodiment provides a wound repair material based on PRF, the wound repair material comprises the following raw materials by weight: 80 parts of PRF gel, 50 parts of Mn-MBG / quercetin composite powder, 0.5 parts of genipin;

[0029] The Mn-MBG / quercetin composite powder comprises the following raw materials in mass ratio: MBG powder: MnCl2·4H2O: quercetin = 1:0.4:0.05;

[0030] The preparation method of the Mn-MBG / quercetin composite powder comprises the following steps:

[0031] (1) MnCl2·4H2O is weighed and dissolved in deionized water to prepare a 0.1 mol / L solution, the pH is adjusted to 5.5 with acetic acid, MBG powder is added, constant temperature magnetic stirring, continuous stirring at 300 rpm for 24 h at 60 ℃, then centrifugation at 10000 rpm for 10 min, washing with ultrapure water and anhydrous ethanol alternately for 3 times, vacuum drying at 60 ℃ for 12 h, to obtain a mixed powder;

[0032] (2) The mixed powder is placed in a muffle furnace for calcination, heated to 600 ℃ at a heating rate of 2 ℃ / min under air atmosphere at room temperature, and kept at this temperature for 2 h to obtain Mn-MBG powder;

[0033] (3) Quercetin is weighed and dissolved in anhydrous ethanol to prepare a stock solution with a concentration of 1.0 mg / mL, and the operation is carried out in the dark, Mn-MBG powder is added to obtain a suspension, the suspension is placed in a light-proof container and sealed with sealing film, and the mixture is placed in a shaking bed at room temperature and in the dark at a low speed of 100 rpm for 24 h to obtain a mixed product;

[0034] (4) centrifuging the mixed product at a speed of 10,000 rpm for 10 min, collecting the solid product, rinsing the precipitate once with a small amount of ice-cold anhydrous ethanol to remove the physically adsorbed quercetin on the surface, transferring the washed product to a culture dish, placing it in a vacuum desiccator, drying at room temperature for 24 h in the dark until completely dry, and obtaining the final Mn-MBG / quercetin composite powder.

[0035] The method for preparing the PRF gel comprises the following steps:

[0036] (a) obtaining a venous blood sample, immediately injecting it into a vacuum blood collection tube containing no anticoagulant, placing the vacuum blood collection tube in a centrifuge, using an optimized gradient centrifugation method, first centrifuging at 700 g for 3 min, and then centrifuging at 300 g for 8 min at room temperature to obtain a centrifuged product;

[0037] (b) carefully taking out the middle amber PRF clot in the centrifuged product with sterile tweezers, placing it in a sterile culture dish, gently rinsing it with normal saline, and carefully removing the red blood cell layer adhered to the bottom, transferring the pure PRF clot to a sterile beaker, and using 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 for 3 times.

[0038] The embodiment also provides a preparation method of a PRF-based wound repair material, which specifically comprises the following steps:

[0039] S1, weighing the Mn-MBG / quercetin composite powder into the PRF gel, adding ice-cold PBS buffer, and magnetically stirring at a speed of 500 rpm in the dark for 45 min to form a uniform and viscous composite slurry;

[0040] S2, injecting the composite slurry into a sterile mold, leveling the surface with a sterile spatula, gently shaking the mold to remove large air bubbles, placing the sterile mold in a 4°C refrigerator for 1 h to allow the fibrinogen of the PRF to further polymerize and be initially shaped, taking it out and quickly transferring it to a-80°C ultra-low temperature refrigerator for rapid freezing for 6 h until complete freezing to form a frozen sample;

[0041] S3, quickly moving the frozen sample into the sample compartment of a freeze dryer, and freeze-drying at-55°C 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;

[0042] S4, weigh genipin, dissolve in PBS buffer with pH 7.4, stir in the dark until completely dissolved, form a genipin cross-linking solution with a mass-volume concentration of 0.2%, immerse the porous sponge body in the genipin cross-linking solution, ensure complete immersion, cross-link at 37°C in the dark for 12 h, after cross-linking is completed, wash thoroughly with ultrapure water to completely remove unreacted genipin, freeze-dry again for 24 h at a temperature of -55°C and a vacuum degree of 0.05 mbar, to obtain a PRF-based wound repair material.

[0043] Example 2: The present example provides a PRF-based wound repair material, which comprises the following raw materials by weight: 90 parts of PRF gel, 100 parts of Mn-MBG / quercetin composite powder, and 1.2 parts of genipin.

[0044] The Mn-MBG / quercetin composite powder comprises the following raw materials in a mass ratio: MBG powder: MnCl2·4H2O: quercetin = 1:0.5:0.08;

[0045] The preparation method of the Mn-MBG / quercetin composite powder comprises the following steps:

[0046] (1) Weigh MnCl2·4H2O, dissolve in deionized water to prepare a 0.2 mol / L solution, adjust the pH to 5.5 with acetic acid, add MBG powder, constant temperature magnetic stirring, continuously stir at 300 rpm for 24 h at 60°C, then centrifuge at 10,000 rpm for 10 min, wash with ultrapure water and anhydrous ethanol alternately for 3 times, vacuum dry at 60°C for 12 h, to obtain a mixed powder;

[0047] (2) Place the mixed powder in a muffle furnace for calcination, heat to 600°C at a heating rate of 3°C / min under air atmosphere at room temperature, and keep the temperature for 2 h to obtain Mn-MBG powder;

[0048] (3) Weigh quercetin, dissolve in anhydrous ethanol to prepare a stock solution with a concentration of 1.5 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 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;

[0049] (4) The mixed product is centrifuged at a speed of 10,000 rpm for 10 min, and the solid product is collected, the precipitate is quickly rinsed twice with a small amount of ice-cold anhydrous ethanol to remove the physically adsorbed quercetin on the surface, the washed product is transferred to a culture dish, and placed in a vacuum desiccator, avoiding light, and dried at room temperature for 24 h until completely dry, to obtain the final Mn-MBG / quercetin composite powder.

[0050] The method for preparing the PRF gel comprises the following steps:

[0051] (a) A venous blood sample is obtained and immediately injected into a vacuum blood collection tube containing no anticoagulant, and the vacuum blood collection tube is placed in a centrifuge, and an optimized gradient centrifugation method is used, 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;

[0052] (b) The centrifuged product is carefully taken out with sterile tweezers, and the middle amber PRF clot is gently washed with normal saline, and the red blood cell layer adhered to the bottom is carefully removed, and the pure PRF clot is transferred to a sterile beaker, and a homogenizer is used to process it into a uniform and viscous PRF gel under ice bath conditions at a speed of 1000 rpm, intermittent operation, 10 s each time, repeated 4 times.

[0053] The embodiment also provides a preparation method of a PRF-based wound repair material, which specifically comprises the following steps:

[0054] S1, Mn-MBG / quercetin composite powder is weighed and added to PRF gel, ice PBS buffer is added, and magnetic stirring is performed at a speed of 500 rpm under ice bath conditions for 45 min to form a uniform and viscous composite slurry;

[0055] S2, the composite slurry is injected into a sterile mold, the surface is scraped flat with a sterile spatula, and the mold is gently shaken to remove large bubbles, and the sterile mold is placed in a 4°C refrigerator for 1 h to allow the fibrinogen of PRF to further polymerize and be preliminarily shaped, and then taken out and quickly transferred to a-80°C ultra-low temperature refrigerator for rapid freezing for 8 h until completely frozen to form a frozen sample;

[0056] S3, the frozen sample is quickly moved into the sample compartment of a freeze dryer, and freeze-dried at-55°C 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;

[0057] S4, weigh genipin, dissolve in PBS buffer with pH 7.4, stir to completely dissolve under light protection, form a genipin cross-linking solution with a mass-volume concentration of 0.25%, immerse the porous sponge body in the genipin cross-linking solution, ensure complete immersion, cross-link at 37°C under light protection for 12 h, after cross-linking is completed, wash with ultrapure water to completely remove unreacted genipin, freeze-dry again for 24 h at a temperature of -55°C and a vacuum degree of 0.05 mbar, to obtain a PRF-based wound repair material.

[0058] Example 3: The present example provides a PRF-based wound repair material, which comprises the following raw materials by weight: 100 parts of PRF gel, 150 parts of Mn-MBG / quercetin composite powder, and 2 parts of genipin.

[0059] 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.

[0060] The preparation method of the Mn-MBG / quercetin composite powder comprises the following steps:

[0061] (1) Weigh MnCl2·4H2O, dissolve in deionized water to prepare a 0.5 mol / L solution, adjust the pH to 6.5 with acetic acid, add MBG powder, constant temperature magnetic stirring, continuously stir at 300 rpm for 24 h at 60°C, then centrifuge at 10,000 rpm for 10 min, wash with ultrapure water and anhydrous ethanol alternately for 3 times, vacuum dry at 60°C for 12 h, to obtain a mixed powder;

[0062] (2) Place the mixed powder in a muffle furnace for calcination, heat to 600°C at a heating rate of 5°C / min under air atmosphere at room temperature, and keep the temperature for 2 h to obtain Mn-MBG powder;

[0063] (3) Weigh quercetin, dissolve in anhydrous ethanol to prepare a stock solution with a concentration of 2.0 mg / mL, operate under light protection, add Mn-MBG powder to obtain a suspension, place the suspension in a light-proof container, seal with sealing film, place in a shaking bed at room temperature and under light protection, and oscillate at a low speed of 100 rpm for 48 h to ensure that quercetin molecules are fully diffused and adsorbed inside the mesoporous channels, to obtain a mixed product;

[0064] (4) centrifuging the mixed product at a speed of 10,000 rpm for 10 min, collecting the solid product, rinsing the precipitate with a small amount of ice-cold anhydrous ethanol twice to remove the physically adsorbed quercetin on the surface, transferring the washed product to a culture dish, placing it in a vacuum desiccator, drying at room temperature for 24 h in the dark until completely dry, and obtaining the final Mn-MBG / quercetin composite powder.

[0065] The method for preparing the PRF gel comprises the following steps:

[0066] (a) obtaining a venous blood sample, immediately injecting it into a vacuum blood collection tube containing no anticoagulant, placing the vacuum blood collection tube in a centrifuge, using an optimized gradient centrifugation method, first centrifuging at 700 g for 3 min, and then centrifuging at 300 g for 8 min at room temperature to obtain a centrifuged product;

[0067] (b) carefully taking out the middle amber PRF clot in the centrifuged product with sterile tweezers, placing it in a sterile culture dish, gently rinsing it with normal saline, and carefully removing the red blood cell layer adhered to the bottom, transferring the pure PRF clot to a sterile beaker, and using 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.

[0068] The embodiment also provides a preparation method of a PRF-based wound repair material, which specifically comprises the following steps:

[0069] S1, weighing the Mn-MBG / quercetin composite powder into the PRF gel, adding ice-cold PBS buffer, and magnetically stirring at a speed of 500 rpm for 45 min in an ice bath to form a uniform and viscous composite slurry;

[0070] S2, injecting the composite slurry into a sterile mold, leveling the surface with a sterile spatula, gently shaking the mold to remove large bubbles, placing the sterile mold in a 4°C refrigerator for 1 h to allow the fibrinogen of the PRF to further polymerize and be initially shaped, taking it out and quickly transferring it to a-80°C ultra-low temperature refrigerator for rapid freezing for 12 h until complete freezing to form a frozen sample;

[0071] S3, quickly moving the frozen sample into the sample compartment of a freeze dryer, and freeze-drying it at-55°C and a vacuum degree of 0.08 mbar for 48 h until all the water in the sample completely sublimates to obtain a primary porous sponge body;

[0072] S4, weigh genipin, dissolve in PBS buffer with pH 7.4, stir to dissolve completely under light protection, form a genipin cross-linking solution with mass concentration of 0.3%, soak the porous sponge body in the genipin cross-linking solution, ensure complete immersion, cross-link at 37°C under light protection for 12 h, after cross-linking is completed, wash with ultrapure water to completely remove unreacted genipin, freeze-dry again for 24 h at a temperature of -55°C and a vacuum degree of 0.08 mbar, and obtain the PRF-based wound repair material.

[0073] The difference between Comparative Example 1 and Example 2 is that no Mn-MBG / quercetin composite powder is added, and the rest is exactly the same as Example 2.

[0074] The difference between Comparative Example 2 and Example 2 is that no MBG is added, and the rest is exactly the same as Example 2.

[0075] The difference between Comparative Example 3 and Example 2 is that no quercetin is added, and the rest is exactly the same as Example 2.

[0076] Experimental Example:

[0077] 1. Cytotoxicity test: MTT method was used for cytotoxicity test, and the wound repair materials prepared by Examples 1-3 and Comparative Examples 1-3 were used as samples, the samples were taken and cut into small pieces of 10 mm x 10 mm x 2 mm, washed with sterile normal saline for 3 times to remove surface residues; in a hundred-level sterile operation table, the samples were placed in a sterile centrifuge tube, 10% fetal bovine serum (FBS) containing DMEM medium was added, sealed and placed in a 37°C, 5% CO2 incubator for static extraction for 24 h to obtain the extraction solution; after extraction, the extraction solution was 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 was prepared as negative control, and medium containing 0.1% Triton X-100 was prepared as positive control). Prepare human skin fibroblasts (HSF) and umbilical vein endothelial cells (HUVEC), and culture in a 37°C, 5% CO2 incubator until the logarithmic growth phase with DMEM medium containing 10% FBS and 1% penicillin-streptomycin; digest the cells with 0.25% trypsin, adjust the cell concentration to 5×10 4 6 / L for standby use. 100 μL of MTT solution was added to each well of the 96-well plate, and incubated for 4 h, then the liquid in the wells was discarded, and dimethyl sulfoxide was added to dissolve the formazan crystals, and the absorbance value (OD value) was measured at 570 nm wavelength by an enzyme-labeled instrument. Calculate the cell survival rate, cell survival rate (%) = (A test group-A blank group) / (A negative control group-A blank group) x 100%), and the results are recorded in Table 1.

[0078] 2. Mechanical property test: The wound repair materials prepared in Examples 1-3 and Comparative Examples 1-3 were used as samples to test the compressive strength. The samples were cut into standard pieces 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 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. Three average values were taken, and the results were recorded 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 on the testing machine clamp to ensure that the sample axis was consistent with the tensile direction. The test was started, and the machine stretched the sample to break, automatically recording the tensile force-displacement curve. The maximum tensile force at break was read from the curve, and the tensile strength was calculated as maximum tensile force / sample parallel section area. The data from the elastic stage (initial linear segment) of the curve were taken, and the elastic modulus was calculated as stress change / strain change. Three average values were taken, and the results were recorded in Table 1.

[0079] Table 1: Cell survival rate and mechanical property test results

[0080]

[0081] As shown in the results of 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 had 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 wound and other application scenarios.

[0082] 3. Antibacterial property: The wound repair materials prepared in Examples 1-3 and Comparative Examples 1-3 were used as samples to test the antibacterial rate by plate counting method. Staphylococcus aureus and Escherichia coli, common pathogenic bacteria for 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 (bacterial solution concentration was adjusted to 1 x 10 6CFU / mL); the sterilized sample (cut into 1 cm x 1 cm small pieces) was added with the bacterial solution at a ratio of sample:bacterial solution = 1 g:10 mL, a blank control group (only bacterial solution) and a sterile dressing control group (medical gauze + bacterial solution) were set up, each group had 3 parallel samples, and the samples were incubated at 37°C for 24 h. After incubation, 1 mL of bacterial solution was taken from each group and gradiently diluted (10 -5 times), 100 μL of the diluted solution was uniformly coated on LB solid culture medium plates, and the plate bacterial colony count (CFU) was counted after 18 h of culture at 37°C. The antibacterial rate of each group was calculated according to the formula: antibacterial rate (%) = (average bacterial colony count of the control group - average bacterial colony count of the test group) / average bacterial colony count of the control group x 100%, and the results were recorded in Table 2.

[0083] 4. Wound healing rate: the wound repair materials prepared by Examples 1-3 and Comparative Examples 1-3 were used as samples, healthy SD rats were selected, a diabetic model was constructed by intraperitoneal injection of streptozotocin (STZ) (blood glucose ≥ 16.7 mmol / L was considered as successful modeling), and the rats were stably fed for 1 week. The rats were randomly divided into a test group, a blank control group (without material application), and a common dressing control group (with medical gauze application), each group had at least 6 rats. After intraperitoneal injection of anesthetic, the back hair of the rats was shaved and disinfected, a sterile surgical ring (diameter 1 cm) was used for positioning, and full-thickness skin (deep to the fascia) was removed to form a standard wound (initial area about 0.785 cm 2 ). The test group was applied with the sterilized sample material, each group was covered with gauze and fixed with tape, and then single-caged feeding was performed. On the 3rd day, 7th day, 14th day, and 21st day after modeling, the rats were lightly anesthetized, the dressing was removed, and the wound photos were taken under uniform conditions using a digital camera with a transparent ruler. After taking the photos, the corresponding materials / dressings were replaced and disinfected. The remaining wound area was measured after calibrating the ruler using ImageJ software, and the average healing rate of each group was calculated according to the formula: wound healing rate (%) = (initial wound area - remaining wound area) / initial wound area x 100%, and the results were recorded in Table 2.

[0084] Table 2: Results of antibacterial property and wound healing rate

[0085]

[0086] 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.

[0087] 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 at 7 days and 14 days, and accelerate the wound healing process. 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 expression of the inflammation genes IL-6 and TNF-α is 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.

[0088] 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 effective support in multiple dimensions 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.

[0089] 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; 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; The preparation method of the PRF-based wound repair material specifically includes the following steps: 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.

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. A method for preparing a PRF-based wound repair material according to any one of claims 1-2, 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.

4. The method for preparing a PRF-based wound repair material according to claim 3, 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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