Preparation method and application of autologous fibrin glue

Abstract

The invention discloses a preparation method and application of autologous fibrin glue, and belongs to the field of biomedical materials. The preparation method comprises the following five steps: collecting autologous venous blood, adding a sodium citrate anticoagulant with the volume ratio of 1: (8-12), and storing at 4-6 DEG C for not more than 2 hours; performing step-by-step centrifugation at the speed of 1500 to 2000 r / min and the speed of 3500 to 4000 r / min to obtain a fibrinogen concentrate; the thrombin-calcium chloride activation solution A and the tranexamic acid activation solution B are mixed according to the ratio of 1: (2-3) to form a composite activation system; mixing the two components according to a mass ratio of 1: (0.8-1.2), and reacting at 25-37 DEG C for 3-5 minutes to form colloid; and pre-freezing at-40 to-60 DEG C, performing vacuum freeze-drying, and storing at 4-8 DEG C. During application, the colloid can be coated on an operation wound surface after being redissolved, or mixed with a growth factor to prepare a dressing for covering a chronic wound surface. The colloid is high in purity, stable in coagulation time, long in storage period, suitable for scenes such as surgical hemostasis and the like, and high in clinical value.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical materials, in particular to a preparation method of autologous blood fibrin glue and application thereof. BACKGROUND

[0002] Autologous blood fibrin glue is a biological glue prepared from the patient's own blood, which has good biocompatibility, degradability and tissue adhesion, and is widely used in clinical for hemostasis, wound closure, and promotion of tissue healing. Compared with biological glue from allogeneic or xenogeneic sources, autologous blood fibrin glue can avoid immune rejection and cross-infection risk, and is particularly suitable for patients sensitive to allogeneic materials or with low immune function.

[0003] However, the traditional preparation method of autologous blood fibrin glue has the following technical defects:

[0004] Low purity and activity: The traditional centrifugal process usually uses single speed centrifugation, which is difficult to effectively separate the impurities (such as red blood cell fragments, platelet debris) in the plasma, resulting in low fibrinogen purity, and further affecting the mechanical properties and hemostatic effect after gelation; at the same time, improper temperature control during centrifugation can easily lead to the inactivation of thrombin, further reducing the activity of the glue.

[0005] Unstable coagulation time: The traditional method usually only uses thrombin-calcium chloride system as an activator, and the coagulation time is greatly affected by environmental temperature and individual differences in blood composition, often resulting in problems such as too fast coagulation (difficult to evenly spread) or too slow coagulation (delayed hemostasis) in clinical, affecting the efficiency and effectiveness of the operation.

[0006] Short storage period: The prepared autologous blood fibrin glue is usually in liquid or semi-solid state, which needs to be stored at low temperature for a short period of time (usually no more than 24 hours), which cannot meet the demand of "one preparation, multiple use" or "preoperative preparation, intraoperative use" in clinical, limiting its popularization and application in primary medical institutions.

[0007] Single application scenario: The traditional autologous blood fibrin glue is mainly used for surface hemostasis, and lacks targeted functional optimization in complex scenarios such as deep soft tissue repair and chronic wound (such as diabetic foot ulcer) healing, with limited repair effect.

[0008] Therefore, it is a technical problem to be solved in the field to develop a preparation method of autologous blood fibrin glue with high purity, controllable coagulation time, long storage period and wide application scenarios. SUMMARY

[0009] The present application aims to provide a preparation method of autologous blood fibrin glue and application thereof to solve the problems raised in the background.

[0010] To achieve the above object, the present application provides the following technical scheme: a preparation method of autologous blood fibrin glue, comprising the following steps:

[0011] Step 1, raw material collection: collect autologous venous blood of a patient, add an anticoagulant sodium citrate solution, the volume ratio of the anticoagulant to the blood is 1:(8-12), shake well and store in a 4-6℃ environment, the storage time is not more than 2 hours;

[0012] Step 2, centrifugal separation: centrifuge the blood treated in step 1 at a speed of 1500-2000r / min for 10-15 minutes to collect the upper plasma; centrifuge the upper plasma at a speed of 3500-4000r / min for 25-30 minutes to remove the supernatant and obtain a fibrinogen concentrate;

[0013] Step 3, preparation of a composite activation system: mix thrombin and a calcium chloride solution to obtain an activation liquid A with a thrombin concentration of 500-1000IU / mL and a calcium chloride concentration of 20-30mmol / L; mix tranexamic acid and physiological saline to obtain an activation liquid B with a tranexamic acid concentration of 5-10mg / mL; mix the activation liquid A and the activation liquid B according to a volume ratio of 1:(2-3) to prepare a composite activation system;

[0014] Step 4, glue preparation: mix the fibrinogen concentrate obtained in step 2 and the composite activation system prepared in step 3 according to a mass ratio of 1:(0.8-1.2), react at 25-37℃ for 3-5 minutes to form autologous blood fibrin glue;

[0015] Step 5, freeze-drying preservation: place the autologous blood fibrin glue prepared in step 4 in a-40 to-60℃ environment for pre-freezing for 2-3 hours, then freeze-dry under a vacuum degree of 0.01-0.03mbar and a temperature of-20 to-10℃ for 8-10 hours, seal and store in a 4-8℃ environment.

[0016] Preferably, the mass concentration of the sodium citrate solution in step 1 is 3.8-4.2%, and the volume ratio of the anticoagulant to the blood is 1:10.

[0017] Preferably, the centrifugal separation in step 2 is implemented according to the following specific step logic:

[0018] Step 2.1, preparation before centrifugation: first, take the autologous venous blood sample with added sodium citrate anticoagulant from the 4-6℃ refrigerated environment, check if there is hemolysis, stratification abnormality, if hemolysis occurs, it needs to be re-collected to avoid the influence of red blood cell fragments on the subsequent fibrinogen purity; then, gently invert the qualified blood sample and shake it for 3-5 times to ensure that the anticoagulant and blood are fully mixed to prevent local blood coagulation; at the same time, start the centrifuge in advance for pre-cooling, adjust the temperature in the centrifuge cavity to 4-6℃, consistent with the storage temperature of the blood sample;

[0019] Step 2.2, first step low-speed centrifugation:

[0020] Centrifugation parameter setting and operation: evenly distribute the shaken blood sample into centrifuge tubes, control the liquid volume in each centrifuge tube to be within 2 / 3 of the tube volume; symmetrically place the centrifuge tubes into the pre-cooled 4-6℃ centrifuge rotor; set the centrifuge speed to 1500-2000r / min, and the centrifugation time to 10-15 minutes;

[0021] Collection of upper plasma after centrifugation: after centrifugation, the centrifuge rotor is completely stopped, the centrifuge tube is slowly taken out, at this time the blood sample has been clearly stratified, from top to bottom, there are light yellow upper plasma, milky white intermediate platelet layer and deep red lower red blood cell and white blood cell layer; using a sterile pipette, slowly insert the pipette along the wall of the centrifuge tube to about 2mm above the interface between the upper plasma and the intermediate platelet layer, avoiding touching the intermediate layer and the lower layer cells, slowly aspirate and transfer the upper plasma to a new sterile centrifuge tube;

[0022] Step 2.3, second step high-speed centrifugation:

[0023] Centrifugation parameter setting and operation: after transferring the collected upper plasma to a new centrifuge tube, check the sealing and balance of the centrifuge tube again to ensure no leakage; set the centrifuge speed to 3500-4000r / min, and the centrifugation time to 25-30 minutes;

[0024] Obtaining fibrinogen concentrate after centrifugation: after centrifugation, the centrifuge tube is taken out, at this time the liquid in the tube presents a clear two-layer structure, the upper layer is light yellow supernatant, and the lower layer is milky white precipitate, which is fibrinogen concentrate; using a sterile pipette, slowly aspirate the upper supernatant, avoiding touching the lower precipitate during the aspiration process, until the supernatant is basically aspirated, only the lower fibrinogen concentrate is left;

[0025] Step 2.4, cleaning and subsequent connection after centrifugal separation: after the completion of the centrifugal separation step, the used centrifuge tube, pipette tip experimental apparatus are strictly treated with sterile to avoid cross contamination; the obtained fibrinogen concentrate is immediately transferred to a sterile operating table, preparing for the step 3 colloidal preparation after the preparation of the complex activation system.

[0026] Preferably, the step 3 of the complex activation system preparation specific process is as follows:

[0027] Step 3.1, raw material preparation and pretreatment before the preparation of the complex activation system: first, confirm the state of the fibrinogen concentrate obtained by centrifugal separation in step 2, which needs to be a milky white semi-solid, free of particulate impurities and hemolysis, and needs to be temporarily stored in a low temperature environment of 4-6℃; secondly, complete the pretreatment and purity verification of the raw materials of the activation system, among which the thrombin needs to be selected from medical grade freeze-dried powder with activity ≥2000IU / mg, calcium chloride needs to be selected from analytical pure anhydrous calcium chloride, tranexamic acid needs to be selected from pharmaceutical grade powder, and normal saline is 0.9% sterile sodium chloride solution. The above raw materials need to be balanced at room temperature for 30 minutes;

[0028] Step 3.2, preparation of activation solution A; the concentration of thrombin in activation solution A is set to 500-1000IU / mL, and the concentration of calcium chloride is 20-30mmol / L; specifically:

[0029] Calcium chloride solution preparation: take 100mL of sterile normal saline, add 0.222-0.333g of anhydrous calcium chloride, and slowly stir with a sterile glass rod until completely dissolved, controlling the stirring speed ≤50r / min during the period to prevent excessive bubbles from affecting the subsequent mixing; after dissolution, the pH value of the solution is measured with a precision pH meter, which needs to be controlled at 6.8-7.2. If the pH value deviates, adjust it by adding 0.1mol / L hydrochloric acid or sodium hydroxide solution;

[0030] Thrombin dissolution and mixing: according to the target concentration, weigh the corresponding mass of thrombin freeze-dried powder, and slowly add it to the above calcium chloride solution. Use a sterile pipette to gently blow and suck 5-8 times to avoid violent shaking that causes thrombin denaturation, until the freeze-dried powder is completely dissolved. The dissolution process is carried out at room temperature, and the dissolution time is controlled within 5 minutes to prevent the thrombin from being exposed to the solution for a long time, which may cause the activity to decrease. After the dissolution is completed, immediately use the thrombin activity determination kit to verify the activity to ensure that the actual activity deviates from the target concentration by ≤5%. If the deviation is too large, it needs to be prepared again;

[0031] Step 3.3, preparation of activation solution B: the concentration of tranexamic acid in activation solution B is set to 5-10mg / mL, which is used as an antifibrinolytic component, and the specific preparation is as follows:

[0032] Tranexamic acid dissolution: Take 100 mL of sterile normal saline, add 0.5-1.0 g of tranexamic acid powder, and place it in a 37°C constant temperature water bath for slow dissolution. Shake gently every 5 minutes during the process until the powder is completely dissolved. The dissolution time should be controlled within 15 minutes.

[0033] Sterile filtration and verification: After dissolution, the tranexamic acid solution is filtered through a 0.22 μm sterile filter under reduced pressure to remove possible microorganisms and impurity particles, ensuring the sterility of the activation solution B. After filtration, 1 mL of filtrate is inoculated into nutrient agar medium and cultured at 37°C for 24 hours. No bacterial growth indicates sterility qualification. If colonies appear, the preparation needs to be re-prepared and the source of contamination needs to be checked.

[0034] Step 3.4, mixed preparation and quality check of the composite activation system:

[0035] Volume ratio control and mixing process: Mix activation solution A and activation solution B according to the volume ratio of 1:(2-3). This ratio is based on the design of "balancing coagulation speed and antifibrinolytic effect" in the invention content. If the proportion of activation solution A is too high (such as 1:1), the coagulation speed is too fast and the antifibrinolytic effect is weak. If the proportion of activation solution A is too low (such as 1:4), the coagulation speed is too slow and the thrombin activity is insufficient. When mixing, use a sterile syringe to extract activation solution A and slowly inject it into a sterile container containing activation solution B. The injection speed is controlled at 1 mL / s, and a sterile magnetic stirrer is used for continuous stirring for 2 minutes to ensure that the two liquids are completely mixed and form a uniform transparent composite activation system, avoiding stratification or local concentration differences.

[0036] Quality detection after mixing: Coagulation time pre-test: Take 0.5 mL of the mixed composite activation system and mix it with 0.5 g of the fibrinogen concentrate obtained in step 2 at 37°C. Use a stopwatch to record the time from mixing to forming a non-flowing colloid. The time should be controlled within 3-5 minutes. If the time deviates, adjust the volume ratio of activation solution A and B. Stability test: Place the composite activation system in a 25°C environment for 30 minutes. Observe for no precipitation, discoloration, or bubble generation. At the same time, measure the pH value of 0.1 mL of the system. It should be maintained at 7.0-7.4 to ensure that the colloid performance is not affected by abnormal pH during the reaction with fibrinogen.

[0037] Step 3.5, connection with subsequent colloid preparation steps: After the preparation of the composite activation system is completed, it needs to be immediately transferred to a sterile operation table and mixed with the fibrinogen concentrate of step 2 according to the mass ratio of 1:(0.8-1.2).

[0038] Preferably, the pre-freezing temperature in step 5 is -50°C, the time is 2.5 hours, the freeze-drying vacuum degree is 0.02 mbar, the temperature is -15°C, and the time is 9 hours.

[0039] Preferably, the application of the preparation method of autologous blood fibrin glue is that the freeze-dried autologous blood fibrin glue is reconstituted with normal saline and then directly applied or sprayed on the surgical wound surface in an amount of 0.5-2 mL / cm 2 Wound surface.

[0040] Preferably, the application of the preparation method of autologous blood fibrin glue is that the autologous blood fibrin glue is mixed with epidermal growth factor or fibroblast growth factor at a mass ratio of 100:(1-5) to prepare a gel dressing, which is applied to the surface of a chronic wound and replaced every 2-3 days.

[0041] Compared with the prior art, the application has the following beneficial effects:

[0042] High purity and strong activity: Through the two-step fractional centrifugation process, the purity of fibrinogen is increased to more than 90%, and the colloid mechanics performance and hemostatic effect are significantly better than those of traditional products.

[0043] Controllable coagulation time: The composite activation system stably controls the coagulation time to be 3-5 minutes, which meets the operation requirements and avoids the problems of too fast or too slow coagulation.

[0044] Long storage period: The storage period after freeze-drying is extended to 6 months, which is convenient for preoperative preparation and batch use, and reduces the cost of clinical application.

[0045] Wide application scenarios: According to different clinical requirements, products suitable for surgical hemostasis, soft tissue repair, and chronic wound healing can be prepared, which are especially suitable for postoperative tumor patients (to avoid the risk of infection of allogeneic materials) and patients with low immune function (to avoid immune rejection), and have strong clinical applicability. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 The figure is a schematic diagram of the method of the application. DETAILED DESCRIPTION

[0047] The technical solutions in the embodiments of the application will be described below in detail with reference to the drawings in the embodiments of the application. Obviously, the described embodiments are only part of the embodiments of the application, not all. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the application.

[0048] Please refer to Figure 1 The application provides a technical solution: a preparation method of autologous blood fibrin glue, comprising the following steps:

[0049] Step 1, raw material collection: collect the patient's autologous venous blood, add an anticoagulant sodium citrate solution, the volume ratio of anticoagulant to blood is 1: (8-12), shake well and store in a 4-6℃ environment, the storage time is not more than 2 hours;

[0050] Step 2, centrifugal separation: centrifuge the blood treated in step 1 at a speed of 1500-2000r / min for 10-15 minutes, collect the upper plasma; centrifuge the upper plasma at a speed of 3500-4000r / min for 25-30 minutes, remove the supernatant, and obtain the fibrinogen concentrate;

[0051] Step 3, preparation of complex activation system: mix thrombin and calcium chloride solution to obtain activation liquid A with a thrombin concentration of 500-1000IU / mL and a calcium chloride concentration of 20-30mmol / L; mix tranexamic acid and normal saline to obtain activation liquid B with a tranexamic acid concentration of 5-10mg / mL; mix activation liquid A and activation liquid B according to a volume ratio of 1: (2-3) to prepare the complex activation system;

[0052] Step 4, colloid preparation: mix the fibrinogen concentrate obtained in step 2 with the complex activation system prepared in step 3 according to a mass ratio of 1: (0.8-1.2), react at 25-37℃ for 3-5 minutes to form autologous fibrin glue;

[0053] Step 5, freeze-drying preservation: place the autologous fibrin glue prepared in step 4 in a -40 to -60℃ environment for 2-3 hours of pre-freezing, then freeze-dry at a vacuum degree of 0.01-0.03mbar and a temperature of -20 to -10℃ for 8-10 hours, seal and store in a 4-8℃ environment.

[0054] Further, the mass concentration of the sodium citrate solution in step 1 is 3.8-4.2%, and the volume ratio of anticoagulant to blood is 1:10.

[0055] Further, the centrifugal separation in step 2 is implemented according to the following specific implementation step logic:

[0056] Step 2.1, preparation before centrifugation: first, take the autologous venous blood sample with added sodium citrate anticoagulant from the 4-6℃ refrigerated environment, check the sample for hemolysis, stratification abnormalities, if hemolysis occurs, re-collection is required to avoid the influence of red blood cell fragments on the purity of subsequent fibrinogen; then, gently invert the qualified blood sample and shake it for 3-5 times to ensure that the anticoagulant and blood are fully mixed to prevent local blood coagulation; at the same time, start the centrifuge in advance for pre-cooling, adjust the temperature in the centrifuge cavity to 4-6℃, consistent with the storage temperature of the blood sample, consistent with the storage temperature of the blood sample, this temperature setting is based on the core requirement of "avoiding protein denaturation" in the invention, which can effectively prevent the inactivation of thrombin and the destruction of fibrinogen structure due to temperature changes during centrifugation, and ensure the subsequent colloid activity;

[0057] Step 2.2, first step low-speed centrifugation:

[0058] Centrifugation parameter setting and operation: evenly distribute the shaken blood sample into centrifuge tubes, control the liquid volume in each centrifuge tube to be within 2 / 3 of the tube volume; symmetrically place the centrifuge tubes into the pre-cooled 4-6℃ centrifuge rotor; set the centrifuge speed to 1500-2000r / min, and the centrifugation time to 10-15 minutes;

[0059] Collection of upper plasma after centrifugation: after centrifugation, the centrifuge rotor is completely stopped, the centrifuge tube is slowly taken out, at this time the blood sample has been clearly stratified, from top to bottom, there are light yellow upper plasma, milky white intermediate platelet layer and deep red lower red blood cell and white blood cell layer; using a sterile pipette, slowly insert the pipette into the centrifuge tube wall to about 2mm above the interface between the upper plasma and the intermediate platelet layer, avoid touching the intermediate layer and the lower layer cells, slowly suck out and transfer the upper plasma to a new sterile centrifuge tube, in this process, the suction speed of the pipette needs to be strictly controlled to prevent the mixing of intermediate platelet into the plasma due to excessive suction, which affects the purity of subsequent fibrinogen. At the same time, record the volume of the collected upper plasma, which provides a reference for the subsequent second step centrifugation operation and the yield estimation of fibrinogen concentrate;

[0060] Step 2.3, second step high-speed centrifugation:

[0061] The centrifugal parameter setting is based on the operation: after the collected upper plasma is transferred to a new centrifugal tube, the sealing and balance of the centrifugal tube are checked again to ensure that there is no liquid leakage; the centrifugal speed is set to 3500-4000 r / min, and the centrifugal time is 25-30 minutes; the setting of this high speed parameter is based on the requirement of "enriching fibrinogen" in the invention content. Compared with the low speed centrifugation in the first step, higher speed can produce greater centrifugal force, so that the fibrinogen with relatively large density in the plasma can be fully settled, and other small molecule proteins (such as albumin, globulin, etc.) in the plasma are retained in the supernatant. At the same time, the temperature in the cavity of the centrifuge still maintains at 4-6℃, which continuously avoids the denaturation of fibrinogen due to temperature rise, and guarantees the biological activity thereof;

[0062] The acquisition of fibrinogen concentrate after centrifugation: after the centrifugation is completed, the centrifugal tube is taken out, at this time the liquid in the tube presents obvious two-layer structure, the upper layer is light yellow supernatant, and the lower layer is cream white precipitate, which is fibrinogen concentrate; the sterile pipette is used to slowly suck the upper supernatant, attention should be paid to avoid touching the lower precipitate during the suction process, until the supernatant is basically sucked, only the lower fibrinogen concentrate is reserved;

[0063] Step 2.4, cleaning and subsequent connection after centrifugal separation: after completing the centrifugal separation step, the used centrifugal tube, pipette tip and experimental apparatus are strictly subjected to sterile treatment to avoid cross contamination; the obtained fibrinogen concentrate is immediately transferred to a sterile operation table, and is prepared to enter the gel preparation link after the preparation of step 3 composite activation system, the whole transfer process needs to be carried out in a sterile environment, and the operation time is controlled within 30 minutes as much as possible, to prevent the concentrate from being contaminated for long time exposure in the air or being affected in activity due to temperature change. At the same time, the parameters in the centrifugal separation process (such as centrifugal speed, time, temperature, plasma volume, concentrate mass, etc.) are recorded to form complete process record, which is convenient for subsequent quality tracing and process optimization, and ensures the rigor and stability of the whole autologous fibrin glue preparation process.

[0064] Further, the specific process of step 3 of preparing composite activation system is as follows:

[0065] Step 3.1, raw material preparation and pretreatment before preparation of composite activation system: first, the state of the fibrinogen concentrate obtained by centrifugal separation in step 2 is confirmed, which needs to be cream white semi-solid, without particulate impurities and hemolysis, and needs to be temporarily stored in a low temperature environment of 4-6℃; second, the pretreatment and purity verification of the raw materials of the activation system are completed, wherein the thrombin needs to select medical grade freeze-dried powder with activity ≥2000 IU / mg, calcium chloride needs to select analytical pure anhydrous calcium chloride, tranexamic acid needs to select pharmaceutical grade powder, and normal saline is 0.9% sterile sodium chloride solution. The above raw materials need to be balanced at room temperature for 30 minutes;

[0066] Step 3.2, Preparation of Activation Liquid A; The concentration of thrombin in Activation Liquid A is set to 500-1000 IU / mL, and the concentration of calcium chloride is 20-30 mmol / L. This parameter range is based on the core requirement of "regulating clotting time with a composite activation system" in the invention. Thrombin, as a direct activator of fibrinogen, has a concentration that is too low, which will cause the clotting time to exceed 5 minutes (unable to meet the requirement of rapid hemostasis during surgery), and a concentration that is too high, which will make the clotting time shorter than 3 minutes (difficult to evenly apply to the wound surface). Calcium chloride, as a synergistic factor for thrombin activity, has a concentration of 20-30 mmol / L to ensure the stability of thrombin conformation. Specifically:

[0067] Calcium chloride solution preparation: Take 100 mL of sterile normal saline, add 0.222-0.333 g of anhydrous calcium chloride, and slowly stir with a sterile glass rod until completely dissolved, controlling the stirring speed ≤ 50 r / min during the process to prevent excessive bubbles that may affect subsequent mixing. After dissolution, use a precision pH meter to measure the pH value of the solution, which needs to be controlled at 6.8-7.2. If the pH value deviates, adjust it by adding 0.1 mol / L hydrochloric acid or sodium hydroxide solution.

[0068] Thrombin dissolution and mixing: According to the target concentration, weigh the corresponding mass of thrombin lyophilized powder and slowly add it to the above calcium chloride solution. Use a sterile pipette to gently blow and suck 5-8 times to avoid vigorous shaking that may cause thrombin denaturation until the lyophilized powder is completely dissolved. The dissolution process is carried out at room temperature and the dissolution time is controlled within 5 minutes to prevent the activity of thrombin from decreasing due to long-term exposure in the solution. After dissolution, immediately use the thrombin activity determination kit for activity verification to ensure that the actual activity deviates ≤ 5% from the target concentration. If the deviation is too large, it needs to be prepared again.

[0069] Step 3.3, Preparation of Activation Liquid B: The concentration of tranexamic acid in Activation Liquid B is set to 5-10 mg / mL, which is used as an antifibrinolytic component. The specific preparation is as follows:

[0070] Tranexamic acid dissolution: Take 100 mL of sterile normal saline and add 0.5-1.0 g of tranexamic acid powder. Place it in a 37°C constant temperature water bath and slowly dissolve it. Shake gently every 5 minutes during the process until the powder is completely dissolved, and the dissolution time is controlled within 15 minutes.

[0071] Sterile filtration and verification: After dissolution, filter the tranexamic acid solution through a 0.22 μm sterile filter under reduced pressure to remove possible microorganisms and impurity particles, ensuring the sterility of Activation Liquid B. After filtration, take 1 mL of filtrate and inoculate it into nutrient agar medium, incubate at 37°C for 24 hours, and observe if there is bacterial growth. If there is bacterial growth, it needs to be prepared again and the source of contamination needs to be investigated.

[0072] Step 3.4, the mixing preparation and quality check of the composite activation system:

[0073] Volume ratio control and mixing process: mix the activation liquid A and the activation liquid B according to the volume ratio of 1: (2-3), which is based on the design of "balancing the coagulation speed and the antifibrinolytic effect" in the invention content: if the proportion of the activation liquid A is too high (such as 1:1), the coagulation speed is too fast and the antifibrinolytic effect is weak; if the proportion of the activation liquid A is too low (such as 1:4), the coagulation speed is too slow and the thrombin activity is insufficient. When mixing, use a sterile syringe to extract the activation liquid A and slowly inject it into a sterile container containing the activation liquid B, the injection speed is controlled at 1 mL / s, at the same time, use a sterile magnetic stirrer to continuously stir for 2 minutes, to ensure that the two liquids are completely mixed and a uniform transparent composite activation system is formed, to avoid stratification or local concentration difference;

[0074] Quality detection after mixing: coagulation time pre-test: take 0.5 mL of the mixed composite activation system and mix it with 0.5 g of the fibrinogen concentrate obtained in step 2 in a 37℃ environment, use a stopwatch to record the time from mixing to forming a non-flowing colloid, which should be controlled within 3-5 minutes, if the time deviates, adjust the volume ratio of the activation liquid A and B; stability test: place the composite activation system in a 25℃ environment for 30 minutes, observe whether there is precipitation, discoloration or bubble generation, which indicates stability; at the same time, take 0.1 mL of the system to measure the pH value, which should be maintained at 7.0-7.4, to ensure that the colloid performance will not be affected by abnormal pH when reacting with fibrinogen;

[0075] Step 3.5, the connection with the subsequent colloid preparation step: after the preparation of the composite activation system is completed, it needs to be immediately transferred to a sterile operation table and mixed with the fibrinogen concentrate of step 2 according to the mass ratio of 1: (0.8-1.2).

[0076] Further, the pre-freezing temperature in step 5 is -50℃, the time is 2.5 hours, the freeze-drying vacuum degree is 0.02 mbar, the temperature is -15℃, and the time is 9 hours.

[0077] Further, the application of the preparation method of the autologous blood fibrin glue, the freeze-dried autologous blood fibrin glue is reconstituted with normal saline and directly applied or sprayed on the surgical wound, the dosage is 0.5-2 mL / cm 2 Wound.

[0078] Further, the application of the preparation method of the autologous blood fibrin glue, the autologous blood fibrin glue is mixed with epidermal growth factor or fibroblast growth factor according to the mass ratio of 100: (1-5) to prepare a gel dressing, which is covered on the surface of a chronic wound and replaced every 2-3 days.

[0079] The application discloses a preparation method of autologous blood fibrin glue and application thereof, and belongs to the field of biomedical materials, and aims to solve the problems of low purity, unstable coagulation time, short storage period and single application scene in traditional preparation methods.

[0080] The preparation method comprises the following five steps: a raw material collecting stage, collecting autologous venous blood and adding sodium citrate anticoagulant with a volume ratio of 1:(8-12), and storing at 4-6 DEG C for no more than 2 hours; a centrifugal separation stage, collecting upper plasma by centrifugation at 1500-2000 r / min for 10-15 minutes, and obtaining fibrinogen concentrate by centrifugation at 3500-4000 r / min for 25-30 minutes; a complex activation system preparation stage, preparing activation liquid A with a thrombin concentration of 500-1000 IU / mL and a calcium chloride concentration of 20-30 mmol / L, and preparing activation liquid B with an amino acid concentration of 5-10 mg / mL, and mixing the two liquids into a complex activation system with a volume ratio of 1:(2-3); a glue preparation stage, mixing the fibrinogen concentrate and the complex activation system with a mass ratio of 1:(0.8-1.2), and reacting at 25-37 DEG C for 3-5 minutes to form glue; and a freeze-drying storage stage, pre-freezing at-40 to-60 DEG C for 2-3 hours, freeze-drying at a vacuum degree of 0.01-0.03 mbar and at-20 to-10 DEG C for 8-10 hours, and storing at 4-8 DEG C after sealing.

[0081] In application, the freeze-dried glue can be reconstituted and applied or sprayed on a surgical wound, or mixed with epidermal growth factor or fibroblast growth factor with a mass ratio of 100:(1-5) to form a dressing for covering a chronic wound.

[0082] Although the embodiments of the application have been shown and described, it is to be understood that for the purpose of the present application, the embodiments can be substantially changed without departing from the spirit and the scope of the present application, the scope of the present application being defined by the appended claims and their equivalents.

Claims

1. A method for preparing autologous fibrin glue, characterized in that: The following steps are involved: Step 1. Raw material collection: Collect the patient's autologous venous blood, add the anticoagulant sodium citrate solution, the volume ratio of the anticoagulant to the blood is 1: (8-12), shake well and store at 4-6 ° C for no more than 2 hours; Step 2, centrifugation: The blood treated in step 1 is centrifuged at 1500-2000 rpm for 10-15 minutes to collect the upper plasma; the upper plasma is then centrifuged at 3500-4000 rpm for 25-30 minutes to remove the supernatant to obtain a fibrinogen concentrate; Step 3, preparation of a composite activation system: mixing thrombin with a calcium chloride solution to obtain an activation solution A having a thrombin concentration of 500-1000 IU / mL and a calcium chloride concentration of 20-30 mmol / L; mixing tranexamic acid with normal saline to obtain an activation solution B having a tranexamic acid concentration of 5-10 mg / mL; and mixing activation solution A and activation solution B in a volume ratio of 1:(2-3) to obtain a composite activation system; Step 4, colloid preparation: the fibrinogen concentrate obtained in step 2 is mixed with the composite activation system prepared in step 3 at a mass ratio of 1:(0.8-1.2), and the mixture is reacted at 25-37°C for 3-5 minutes to form an autologous fibrin glue; Step 5, freeze-drying and storage: pre-freeze the autologous fibrin glue prepared in step 4 at -40 to -60°C for 2-3 hours, then freeze-dry at a vacuum of 0.01-0.03 mbar and a temperature of -20 to -10°C for 8-10 hours, seal and store at 4-8°C.

2. The method for preparing an autologous fibrin glue according to claim 1, characterized in that: The mass concentration of the sodium citrate solution in step 1 is 3.8-4.2%, and the volume ratio of the anticoagulant to the blood is 1:

10.

3. The method for preparing an autologous fibrin glue according to claim 1, characterized in that: The specific implementation steps of the centrifugal separation in step 2 are as follows: Step 2.

1. Preparation before centrifugation: First, remove the autologous venous blood sample containing sodium citrate anticoagulant from the refrigerated environment at 4-6°C and inspect the sample for hemolysis and abnormal stratification. If hemolysis is present, recollect the sample to prevent red blood cell fragments from affecting the subsequent fibrinogen purity. Then, gently shake the qualified blood sample 3-5 times to ensure that the anticoagulant and blood are fully mixed to prevent local blood coagulation. At the same time, start the centrifuge in advance to pre-cool the centrifuge chamber and adjust the temperature of the centrifuge chamber to 4-6°C, consistent with the storage temperature of the blood sample. Step 2.2, first step of low speed centrifugation: Centrifuge parameter setting and operation: Evenly divide the shaken blood sample into centrifuge tubes, and control the liquid volume of each centrifuge tube to be within 2 / 3 of the tube volume; symmetrically place the centrifuge tubes in the centrifuge rotor pre-cooled to 4-6°C; set the centrifuge speed to 1500-2000r / min and the centrifugation time to 10-15 minutes; Collection of upper plasma after centrifugation: After centrifugation, wait until the centrifuge rotor stops completely and slowly remove the centrifuge tube. The blood sample is now clearly separated into layers, from top to bottom: a light yellow upper plasma layer, a milky white middle platelet layer, and a dark red lower red blood cell and white blood cell layer. Use a sterile pipette to slowly insert it along the wall of the centrifuge tube to approximately 2 mm above the interface between the upper plasma layer and the middle platelet layer, avoiding touching the middle and lower cell layers. Slowly aspirate the upper plasma layer and transfer it to a new sterile centrifuge tube. Step 2.3, second high-speed centrifugation: Centrifuge parameter setting basis and operation: After transferring the collected upper plasma to a new centrifuge tube, check the sealing and balance of the centrifuge tube again to ensure there is no leakage; set the centrifuge speed to 3500-4000r / min and the centrifugation time to 25-30 minutes; Obtaining fibrinogen concentrate after centrifugation: After centrifugation, remove the centrifuge tube. The liquid in the tube now has a distinct two-layer structure: the upper layer is a light yellow supernatant, and the lower layer is a milky white precipitate, which is the fibrinogen concentrate. Use a sterile pipette to slowly aspirate the upper supernatant, taking care to avoid touching the lower precipitate during aspiration, until the supernatant is almost completely aspirated, leaving only the fibrinogen concentrate in the lower layer. Step 2.4, post-centrifugation cleaning and subsequent connection: After completing the centrifugation step, strictly sterilize the used centrifuge tubes and pipette tips to avoid cross contamination; immediately transfer the obtained fibrinogen concentrate to a sterile operating table and prepare to enter the colloid preparation link after the preparation of the composite activation system in step 3.

4. The method for preparing an autologous fibrin glue according to claim 1, characterized in that: The specific process for preparing the composite activation system in step 3 is as follows: Step 3.

1. Preparation and pretreatment of raw materials before preparation of the composite activation system: First, confirm the state of the fibrinogen concentrate obtained by centrifugation in Step 2. It must be a milky white semi-solid, free of particulate impurities and hemolysis, and must be temporarily stored at a low temperature of 4-6°C. Next, complete the pretreatment and purity verification of the activation system raw materials. For thrombin, use medical-grade lyophilized powder with an activity of ≥2000 IU / mg, analytically pure anhydrous calcium chloride, and pharmaceutical-grade powdered tranexamic acid. The physiological saline should be a 0.9% sterile sodium chloride solution. All of the above raw materials must be equilibrated at room temperature for 30 minutes. Step 3.2, preparation of activation solution A; the thrombin concentration in activation solution A is set to 500-1000 IU / mL, and the calcium chloride concentration is set to 20-30 mmol / L; specifically: Preparation of calcium chloride solution: Take 100 mL of sterile normal saline, add 0.222-0.333 g of anhydrous calcium chloride, and slowly stir with a sterile glass rod until completely dissolved. During this period, control the stirring speed to ≤50 r / min to prevent excessive bubbles from affecting subsequent mixing. After dissolution is complete, use a precision pH meter to measure the pH value of the solution, which should be controlled between 6.8 and 7.

2. If the pH value deviates, fine-tune it by adding 0.1 mol / L hydrochloric acid or sodium hydroxide solution. Dissolving and mixing thrombin: Weigh the corresponding mass of lyophilized thrombin powder according to the target concentration and slowly add it to the above-mentioned calcium chloride solution. Use a sterile pipette to gently pipette 5-8 times to avoid violent shaking that may cause denaturation of thrombin, until the lyophilized powder is completely dissolved. The dissolution process should be carried out at room temperature and the dissolution time should be controlled within 5 minutes to prevent the activity of thrombin from being exposed to the solution for a long time and thus decreasing. After the dissolution is completed, immediately use the thrombin activity assay kit to check the activity to ensure that the deviation between the actual activity and the target concentration is ≤5%. If the deviation is too large, re-preparation is required. Step 3.3, Preparation of Activation Solution B: The concentration of tranexamic acid in Activation Solution B is set to 5-10 mg / mL, which is used as an antifibrinolytic component. The specific preparation is as follows: Dissolution of tranexamic acid: Take 100 mL of sterile saline, add 0.5-1.0 g of tranexamic acid powder, place in a 37°C constant temperature water bath and slowly dissolve, gently shaking every 5 minutes until the powder is completely dissolved. The dissolution time should be controlled within 15 minutes. Sterile filtration and verification: After dissolution is complete, filter the tranexamic acid solution through a 0.22 μm sterile filter membrane under reduced pressure to remove any microorganisms and impurities to ensure the sterility of Activation Solution B. After filtration, take 1 mL of the filtrate and inoculate it into nutrient agar medium. Incubate at 37°C for 24 hours. If no bacterial growth is observed, the solution is sterile. If colonies are present, the solution must be re-prepared and the source of contamination must be investigated. Step 3.4: Mixing preparation and quality verification of the composite activation system: Volume ratio control and mixing process: Mix Activation Liquid A and Activation Liquid B in a volume ratio of 1:(2-3). During mixing, use a sterile syringe to draw Activation Liquid A and slowly inject it into a sterile container containing Activation Liquid B at a speed of 1 mL / s. Use a sterile magnetic stirrer to stir continuously for 2 minutes to ensure that the two liquids are completely mixed to form a uniform and transparent composite activation system, avoiding stratification or local concentration differences. Post-mixing quality inspection: Pre-clotting time test: Take 0.5mL of the mixed composite activation system and mix it with 0.5g of fibrinogen concentrate obtained in step 2 at 37°C. Use a stopwatch to record the time from mixing to the formation of a non-flowing colloid. It must be controlled within 3-5 minutes. If the time deviates, correct it by fine-tuning the volume ratio of activation liquid A to B. Stability test: Place the composite activation system at 25°C and let it stand for 30 minutes. If there is no precipitation, discoloration, or bubbles, it is stable. At the same time, take 0.1mL of the system to measure the pH value. It must be kept between 7.0 and 7.4 to ensure that the colloid performance is not affected by abnormal pH when reacting with fibrinogen. Step 3.5, connection with subsequent colloid preparation steps: After the composite activation system is prepared, it must be immediately transferred to a sterile operating table and mixed with the fibrinogen concentrate in step 2 at a mass ratio of 1:(0.8-1.2).

5. The method for preparing an autologous fibrin glue according to claim 1, characterized in that: In step 5, the pre-freezing temperature is -50°C and the time is 2.5 hours, and the freeze-drying vacuum is 0.02 mbar, the temperature is -15°C and the time is 9 hours.

6. Use of the method for preparing autologous fibrin glue according to any one of claims 1 to 5, characterized in that: Redissolve the freeze-dried autologous fibrin glue with normal saline and apply or spray it directly on the surgical wound at a dosage of 0.5-2 mL / cm 2 Wound.

7. Use of the method for preparing autologous fibrin glue according to any one of claims 1 to 5, characterized in that: Autologous fibrin glue is mixed with epidermal growth factor or fibroblast growth factor in a mass ratio of 100:(1-5) to make a gel dressing, which is covered on the surface of chronic wounds and replaced every 2-3 days.

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