CN115068685A - Platelet-rich fibrin composite membrane and preparation method and application thereof - Google Patents
Platelet-rich fibrin composite membrane and preparation method and application thereof Download PDFInfo
- Publication number
- CN115068685A CN115068685A CN202210773569.6A CN202210773569A CN115068685A CN 115068685 A CN115068685 A CN 115068685A CN 202210773569 A CN202210773569 A CN 202210773569A CN 115068685 A CN115068685 A CN 115068685A
- Authority
- CN
- China
- Prior art keywords
- platelet
- fat
- rich fibrin
- composite membrane
- nano
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
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Images
Classifications
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Abstract
The invention discloses a platelet-rich fibrin composite membrane and a preparation method and application thereof, wherein the platelet-rich fibrin composite membrane is a nano-fat combined platelet-rich fibrin composite membrane, and the nano-fat combined platelet-rich fibrin composite membrane is prepared from autologous nano-fat, platelet-rich fibrin, quaternized chitosan and medical CaCl 2 The solution and the antioxidant are prepared by ionic crosslinking. The invention belongs to the field of biomedical materials, and provides a nano-fat combined platelet-rich fibrin composite membrane prepared from autologous nano-fat, PRF, QCS, medical CaCl2 solution and an antioxidant. The composite membrane provided by the invention not only can continuously release growth factors on the wound surface of the diabetic foot, but also has good mechanical properties, antibacterial and anti-inflammatory properties, active oxygen scavenging and controllable degradation capability.
Description
Technical Field
The invention belongs to the field of biomedical materials, and particularly relates to a platelet-rich fibrin composite membrane, a preparation method and application thereof, more specifically to application of the platelet-rich fibrin composite membrane to a diabetic foot wound.
Background
The treatment cost of the diabetic foot wound is high and the time consumption is long, so that great burden is caused to a patient, and simultaneously great challenge is brought to a medical system. At present, the methods for treating diabetic foot wounds comprise growth factors, negative pressure wound drainage, ultrasonic therapy, maggot therapy, phototherapy, local debridement treatment, artificial skin substitutes and hyperbaric oxygen therapy, and the treatment effect is improved; however, most patients still have amputation due to the limitation of treatment methods, so that the labor capacity and the self-care ability of life are lost, and the life quality of the patients is greatly reduced. Thus, the clinician's focus in treating diabetic feet is shifting from passive therapy to early intervention and active exploration of more rational treatment regimens.
The diabetic foot wound is difficult to heal because the growth factor at the wound has low content and poor activity, so that characteristic cells cannot be effectively activated and repaired, the proliferation, differentiation and migration of the cells are hindered, the granulation tissue formation and the wound microcirculation reconstruction are inhibited, and the normal healing process of the wound is interrupted. The platelet-rich fibrin (PRF) is a platelet concentrate prepared by centrifuging autologous blood at a low speed and a long time, contains uniformly distributed and wide-range growth factors, has strong antibacterial and anti-inflammatory capabilities and potential for promoting wound tissue healing and regeneration, and is considered to be one of the most promising methods for treating diabetic foot wounds. However, Platelet Rich Fibrin (PRF) also has significant drawbacks: on one hand, the blood needs to be taken for many times, and the blood is prepared on the spot when in use, so that serious psychological disorder is caused to a patient; on the other hand, the platelet-rich fibrin (PRF) gel has the problems of short action time of growth factors, easy loss and the like, and the two points limit the clinical application of the platelet-rich fibrin gel to a certain extent.
Disclosure of Invention
Aiming at the situation, aiming at solving the problems that the platelet-rich fibrin (PRF) has multiple blood sampling, the existing preparation and the short action time and easy loss of the growth factor in the traditional platelet-rich fibrin (PRF) gel when the platelet-rich fibrin (PRF) is used for treating diabetic foot wounds, the invention provides a platelet-rich fibrin composite membrane and a preparation method and application thereof, wherein the platelet-rich fibrin composite membrane adopted in the invention is a nano fat combined platelet-rich fibrin composite membrane, the preparation method of the nano fat combined platelet-rich fibrin composite membrane is simple, the nano fat combined platelet-rich fibrin composite membrane can be stored for a long time under mild conditions, the psychological loss of patients caused by multiple blood sampling and existing preparation is avoided, and the prepared platelet-rich fibrin composite membrane has a loose fibrin structure, high content of platelets, leukocytes and growth factors, and is uniform and wide in distribution; and secondly, the foot wound surface protecting agent has strong antibacterial and anti-inflammatory capabilities and active oxygen removing capability, and further solves the problems of growth factor deficiency, growth factor activity reduction and the like caused by susceptibility and excessive active oxygen at the foot wound surface of the diabetic.
The invention provides the following technical scheme: the platelet-rich fibrin composite membrane is a nano-fat combined platelet-rich fibrin composite membrane, and is prepared from autologous nano-fat, platelet-rich fibrin (PRF), Quaternized Chitosan (QCS) and medical CaCl 2 The solution and the antioxidant are prepared by ion crosslinking.
Further, the nano-meterThe volume ratio of the raw materials of the formula of the fat-combined platelet-rich fibrin composite membrane is as follows: the volume ratio of the platelet-rich fibrin (PRF) to the autologous nano fat is 1: 5-1: 3, the volume ratio of the platelet-rich fibrin (PRF) to the Quaternized Chitosan (QCS) is 1: 5-1: 1, the volume ratio of the platelet-rich fibrin (PRF) to the antioxidant is 1: 3-1: 1, and medical CaCl is added into each milliliter of the platelet-rich fibrin (PRF) 2 The amount of the solution is 200-500. mu.L/mL.
Further, the mass fraction of the Quaternized Chitosan (QCS) is 2-6%.
Further, the antioxidant is selected from one or a mixture of more than two of gallic acid, tannic acid, tea yellow pigment, dopamine, resveratrol, sodium phytate and vitamin C.
Further, the mass fraction of the antioxidant is 1-4%.
The invention provides a preparation method of a platelet-rich fibrin composite membrane, which specifically comprises the following steps:
s1, mixing the autologous nano fat and the platelet-rich fibrin (PRF) uniformly according to the volume ratio of the raw materials of the formula, and placing the mixture at 4 ℃ for later use;
s2, quaternizing the chitosan (QCS), the antioxidant and the medical CaCl under the normal temperature condition 2 The solution is stirred in proportion to form uniform mixed solution;
s3, stirring the mixture obtained in the step S1 and the mixed solution obtained in the step S2 uniformly at 4 ℃, transferring the mixture to a culture dish, and standing to form a membrane.
Further, the preparation method of the autologous nano-fat in step S1 is as follows:
(1) performing local swelling anesthesia around the navel of the abdomen of a patient, connecting a 20mL syringe with a liposuction needle with the aperture of 1mm, and performing radial suction under the condition of artificial negative pressure to obtain about 100mL of tiny granular fat;
(2) standing the injector for 10-15min, obviously layering the tiny fat particles and the swelling solution, removing the swelling solution, and washing the tiny fat particles with normal saline for 2-3 times;
(3) distributing the tiny particle fat in the step (2) into a 20mL injector, centrifuging for 3min at the rotating speed of 2000-3000rpm/min, removing water, and collecting the tiny particle fat;
(4) subpackaging the micro-particle fat collected in the step (3) in a 20mL syringe, connecting with a nano fat converter, continuously and mechanically cutting for 10-20 times, mechanically emulsifying, and continuously oscillating the emulsified fat for 1-2min under the condition of artificial negative pressure;
(5) centrifuging at the rotating speed of 2000-;
(6) filtering the emulsified fat with a filter screen with pore diameter of 0.5mm for 2-3 times, and removing the residual connective tissue to obtain fat emulsion, namely the autologous nano fat.
Further, the Platelet Rich Fibrin (PRF) is prepared in step S1 as follows:
(1) collecting 100mL of venous blood of a patient, and putting the collected blood into a centrifuge tube without anticoagulant;
(2) immediately centrifuging at the rotating speed of 1000-1600 rpm/min for 10-20min, and taking the middle gel layer to obtain platelet-rich fibrin (PRF);
(3) the obtained platelet-rich fibrin (PRF) is stored at 4 ℃ for further use.
The invention provides an application of a platelet-rich fibrin composite membrane, in particular to an application of a nano-fat and platelet-rich fibrin composite membrane in diabetic foot wound surfaces, tooth tissue regeneration membranes and artificial skin.
The invention with the structure has the following beneficial effects: compared with the prior art, the platelet-rich fibrin composite membrane and the preparation method and the application thereof provided by the invention have the following advantages:
(1) the nano-fat and platelet-rich fibrin composite membrane provided by the invention adopts autologous nano-fat, PRF, QCS and antioxidant to carry out ionic crosslinking through CaCl2 solution, and the components are synergistic with each other, so that the mechanical property of the composite membrane is improved, and a protective barrier is provided for the wound surface.
(2) The components such as QCS, antioxidant and the like in the nano-fat combined platelet-rich fibrin composite membrane provided by the invention can inhibit bacterial reproduction and remove active oxygen at the wound surface of diabetic foot, and provide a good healing environment for the wound surface.
(3) The autologous nano fat in the nano fat combined platelet-rich fibrin composite membrane provided by the invention is combined with PRF, so that the immunological rejection is reduced; the composite membrane contains main active ingredients such as uniform platelets, growth factors and leukocytes in the wound healing process, can provide the wound with the growth factors with strong activity, multiple types and high concentration, and the growth factors synergistically promote collagen synthesis, angiogenesis, tissue remodeling and re-epithelialization through different regeneration mechanisms, shorten the healing time of the wound, and can effectively solve the problems of lack of the growth factors and activity reduction of chronic wounds.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 is a platelet-rich fibrin composite membrane, a preparation method thereof and autologous nano-fat prepared by the application thereof, according to the present invention;
FIG. 2 is a process diagram of a platelet-rich fibrin composite membrane, a preparation method thereof and a preparation process of platelet-rich fibrin (PRF) using the same;
FIG. 3 is a diagram showing the usage effect of the platelet-rich fibrin composite membrane, the preparation method thereof and the nano-fat combined platelet-rich fibrin composite membrane prepared by the application thereof.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments; all other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It should be noted that the terms "front," "back," "left," "right," "upper" and "lower" used in the following description refer to directions in the drawings, and the terms "inner" and "outer" refer to directions toward and away from, respectively, the geometric center of a particular component.
Example 1
1. Preparing autologous nano fat:
(1) local swelling anesthesia is carried out on the abdominal periphery of the navel of the patient;
(2) after the anesthesia takes effect, a liposuction needle with the aperture of 1mm is connected with a 20mL syringe for standby;
(3) performing radial suction under the condition of artificial negative pressure to obtain about 100mL of tiny particle fat;
(4) standing the injector for 15min, obviously layering the tiny fat particles and the swelling solution, removing the swelling solution, washing the tiny fat particles with normal saline for 3 times, and removing blood cells;
(5) subpackaging the tiny particle fat in a 20mL injector, centrifuging at the rotating speed of 2500rpm/min for 3min, obviously layering the fat and the water, and removing the water;
(6) subpackaging the tiny particle fat in a 20mL syringe, connecting with a nano fat converter, continuously and mechanically cutting for 15 times, mechanically emulsifying, and continuously oscillating the emulsified fat for 1min under the condition of artificial negative pressure;
(7) centrifuging at 2500rpm/min for 3min to obviously separate emulsified fat from oil, pouring off oil, sucking residual oil with gauze, and collecting emulsified fat;
(8) filtering emulsified fat with 0.5mm filter screen for 3 times to remove residual connective tissue to obtain fat emulsion as nanometer fat;
preparation of PRF:
(1) collecting 10 mL/10 venous blood of a patient, and putting the collected blood into a centrifuge tube without anticoagulant;
(2) centrifuging at 1400rpm/min for 20min to divide blood into three layers, including upper layer of platelet-poor plasma layer, middle layer of RPF gel layer, and lowest layer of erythrocyte layer;
(3) storing the centrifuge tube in a refrigerator at 4 deg.C for 30min, discarding the supernatant, and sucking the intermediate layer with a sterile straw to obtain PRF;
3. preparing a nano-fat combined platelet-rich fibrin composite membrane:
s1, uniformly mixing 5mL of autologous nano fat with 1mL of PRF according to a proportion, and placing at 4 ℃ for later use;
s2, under the condition of normal temperature, 5mL QCS (mass fraction: 3%), 3mL tannic acid (mass fraction: 2%) and 400 μ L medical CaCl 2 Stirring the solution to form a uniform mixed solution;
s3, stirring the mixture obtained in the step S1 and the mixed solution of S2 uniformly at 4 ℃, transferring the mixture to a culture dish, and standing to form a membrane.
Example 2
1. Preparing autologous nano fat:
(1) local swelling anesthesia is carried out around the navel of the abdomen of the patient;
(2) after local anesthesia takes effect, a liposuction needle with the aperture of 1mm is connected with a 20mL syringe for standby;
(3) performing radial suction under the condition of artificial negative pressure to obtain about 100mL of tiny particle fat;
(4) standing the injector for 15min, obviously layering the tiny fat particles and the swelling solution, removing the swelling solution, washing the tiny fat particles with normal saline for 3 times, and removing blood cells;
(5) subpackaging the tiny particle fat in a 20mL injector, centrifuging at 3000rpm/min for 3min, obviously layering the fat and the water, and removing the water;
(6) subpackaging the tiny particle fat in a 20mL syringe, connecting with a nano fat converter, continuously and mechanically cutting for 10-20 times, mechanically emulsifying, and continuously oscillating the emulsified fat for 1min under the condition of artificial negative pressure;
(7) centrifuging at 3000rpm/min for 3min to obviously separate emulsified fat from oil, pouring off oil, sucking residual oil with gauze, and collecting emulsified fat;
(8) filtering emulsified fat with 0.5mm filter screen for 3 times to remove residual connective tissue to obtain fat emulsion as nanometer fat;
preparation of PRF:
(1) collecting 10 mL/10 venous blood of a patient, and putting the collected blood into a centrifuge tube without anticoagulant;
(2) centrifuging at 1400rpm/min for 20min to divide blood into three layers, including upper layer of platelet-poor plasma layer, middle layer of RPF gel layer, and lowest layer of erythrocyte layer;
(3) storing the centrifuge tube in a refrigerator at 4 deg.C for 30min, discarding the supernatant, and sucking the intermediate layer with a sterile straw to obtain PRF;
3. preparing a nano-fat combined platelet-rich fibrin composite membrane:
s1, uniformly mixing 5mL of autologous nano fat with 1mL of PRF according to a proportion, and placing at 4 ℃ for later use;
s2, stirring 5mL of QCS (mass fraction: 3%), 3mL of tannic acid (mass fraction: 2%) and 400 mu L of medical CaCl2 at normal temperature to form a uniform mixed solution;
s3, stirring the mixture of the mixture obtained in the step S1 and S2 at 4 ℃, transferring the mixture to a culture dish, and standing to form a film.
Example 3
1. Preparing autologous nano fat:
(1) local swelling anesthesia is carried out around the navel of the abdomen of the patient;
(2) after local anesthesia takes effect, a liposuction needle with the aperture of 1mm is connected with a 20mL syringe for standby;
(3) performing radial suction under the condition of artificial negative pressure to obtain about 100mL of tiny particle fat;
(4) standing the injector for 15min, obviously layering the tiny fat particles and the swelling solution, removing the swelling solution, washing the tiny fat particles with normal saline for 3 times, and removing blood cells;
(5) subpackaging the tiny particle fat in a 20mL injector, centrifuging at the rotating speed of 2500rpm/min for 3min, obviously layering the fat and the water, and removing the water;
(6) subpackaging the tiny particle fat in a 20mL syringe, connecting with a nano fat converter, continuously and mechanically cutting for 10-20 times, mechanically emulsifying, and continuously oscillating the emulsified fat for 1min under the condition of artificial negative pressure;
(7) centrifuging at 2500rpm/min for 3min to obviously separate emulsified fat from oil, pouring off oil, sucking residual oil with gauze, and collecting emulsified fat;
(8) filtering emulsified fat with 0.5mm filter screen for 3 times to remove residual connective tissue to obtain fat emulsion as nanometer fat;
preparation of PRF:
(1) collecting 10 mL/10 venous blood of a patient, and putting the collected blood into a centrifuge tube without anticoagulant;
(2) immediately centrifuging at 1400rpm/min for 20min to divide the blood into three layers, wherein the upper layer is a platelet poor plasma layer, the middle layer is an RPF gel layer, and the lowest layer is a red blood cell layer;
(3) storing the centrifuge tube in a refrigerator at 4 deg.C for 30min, discarding the supernatant, and sucking the intermediate layer with a sterile straw to obtain PRF;
3. preparing a nano-fat combined platelet-rich fibrin composite membrane:
s1, uniformly mixing 10mL of autologous nano fat with 2mL of PRF according to a proportion, and placing at 4 ℃ for later use;
s2, stirring 10mL of QCS (mass fraction: 3%), 6mL of tannic acid (mass fraction: 2%) and 800 mu L of medical CaCl2 at normal temperature to form a uniform mixed solution;
s3, stirring the mixture obtained in the step S1 and the mixed solution of S2 uniformly at 4 ℃, transferring the mixture to a culture dish, and standing to form a membrane.
Example 4
1. Preparing autologous nano fat:
(1) local swelling anesthesia is carried out around the navel of the abdomen of the patient;
(2) after local anesthesia takes effect, a liposuction needle with the aperture of 1mm is connected with a 20mL syringe for standby;
(3) performing radial suction under the condition of artificial negative pressure to obtain about 100mL of tiny particle fat;
(4) standing the injector for 15min, obviously layering the tiny fat particles and the swelling solution, removing the swelling solution, washing the tiny fat particles with normal saline for 3 times, and removing blood cells;
(5) subpackaging the tiny particle fat in a 20mL injector, centrifuging at the rotating speed of 2500rpm/min for 3min, obviously layering the fat and the water, and removing the water;
(6) subpackaging the tiny particle fat in a 20mL syringe, connecting with a nano fat converter, continuously and mechanically cutting for 10-20 times, mechanically emulsifying, and continuously oscillating the emulsified fat for 1min under the condition of artificial negative pressure;
(7) centrifuging at 2500rpm/min for 3min to obviously separate emulsified fat from oil, pouring off oil, sucking residual oil with gauze, and collecting emulsified fat;
(8) filtering emulsified fat with 0.5mm filter screen for 3 times to remove residual connective tissue to obtain fat emulsion as nanometer fat;
preparation of PRF:
(1) collecting 10 mL/10 venous blood of a patient, and putting the collected blood into a centrifuge tube without anticoagulant;
(2) centrifuging at 1500rpm/min for 14min to divide blood into three layers, including platelet-poor plasma layer in the upper layer, RPF gel layer in the middle layer, and erythrocyte layer in the lowest layer;
(3) placing the centrifuge tube in a refrigerator at 4 deg.C for 30min, discarding the supernatant, and sucking the middle layer with a sterile straw to obtain PRF;
3. preparing a nano-fat combined platelet-rich fibrin composite membrane:
s1, uniformly mixing 5mL of autologous nano fat with 1mL of PRF according to a proportion, and placing at 4 ℃ for later use;
s2, stirring 5mL of QCS (mass fraction: 4%), 3mL of resveratrol (mass fraction: 2%) and 400 mu L of medical CaCl2 to form a uniform mixed solution at normal temperature;
s3, stirring the mixture obtained in the step S1 and the mixed solution of S2 uniformly at 4 ℃, transferring the mixture to a culture dish, and standing to form a membrane.
Comparative example 1
In comparison with example 1, no autologous nano-fat was added to comparative example 1.
Preparation of PRF:
(1) collecting 10 mL/10 venous blood of a patient, and placing the collected blood into a centrifuge tube without anticoagulant;
(2) centrifuging at 1500rpm/min for 14min to divide blood into three layers, including platelet-poor plasma layer in the upper layer, RPF gel layer in the middle layer, and erythrocyte layer in the lowest layer;
(3) storing the centrifuge tube in a refrigerator at 4 deg.C for 30min, discarding the supernatant, and sucking the intermediate layer with a sterile straw to obtain PRF;
2. preparing a platelet-rich fibrin composite membrane:
s1, extracting 1mL of PRF, and placing at 4 ℃ for later use;
s2, stirring 5mL of QCS (mass fraction: 4%), 3mL of tannic acid (mass fraction: 2%) and 400 mu L of medical CaCl2 to form a uniform mixed solution at normal temperature;
s3, stirring the mixture obtained in the step S1 and the mixed solution of S2 uniformly at 4 ℃, transferring the mixture to a culture dish, and standing to form a membrane.
Comparative example 2
In comparison with example 1, no QCS and no antioxidant were added in comparative example 2.
1. Preparing autologous nano fat:
(1) local swelling anesthesia is carried out around the navel of the abdomen of the patient;
(2) after local anesthesia takes effect, a liposuction needle with the aperture of 1mm is connected with a 20mL syringe for standby;
(3) performing radial suction under the condition of artificial negative pressure to obtain about 100mL of tiny particle fat;
(4) standing the injector for 15min, obviously layering the tiny fat particles and the swelling solution, removing the swelling solution, washing the tiny fat particles with normal saline for 3 times, and removing blood cells;
(5) subpackaging the tiny particle fat in a 20mL injector, centrifuging at the rotating speed of 2500rpm/min for 3min, obviously layering the fat and the water, and removing the water;
(6) subpackaging the tiny particle fat in a 20mL syringe, connecting with a nano fat converter, continuously and mechanically cutting for 10-20 times, mechanically emulsifying, and continuously oscillating the emulsified fat for 1min under the condition of artificial negative pressure;
(7) centrifuging at 2500rpm/min for 3min to obviously separate emulsified fat from oil, pouring off oil, sucking residual oil with gauze, and collecting emulsified fat;
(8) filtering emulsified fat with 0.5mm filter screen for 3 times to remove residual connective tissue to obtain fat emulsion as nanometer fat;
preparation of PRF:
(1) collecting 10 mL/10 venous blood of a patient, and putting the collected blood into a centrifuge tube without anticoagulant;
(2) centrifuging at 1500rpm/min for 14min to divide blood into three layers, including platelet-poor plasma layer in the upper layer, RPF gel layer in the middle layer, and erythrocyte layer in the lowest layer;
(3) storing the centrifuge tube in a refrigerator at 4 deg.C for 30min, discarding the supernatant, and sucking the intermediate layer with a sterile straw to obtain PRF;
3. preparing a platelet-rich fibrin composite membrane:
s1, uniformly mixing 5mL of autologous nano fat with 1mL of PRF according to a proportion, and placing at 4 ℃ for later use;
s2, stirring uniformly 400 mu L of medical CaCl2 and S1 mixed solution at 4 ℃, transferring the mixed solution into a culture dish, and standing to form a film.
Compared with comparative examples 1 and 2, the composite membrane prepared by the example 1 has better mechanical property, antibacterial and anti-inflammatory capability and active oxygen scavenging capability than those of the comparative examples 1 and 2, and is more suitable for being used on the wound surface of the diabetic foot.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (8)
1. The platelet-rich fibrin composite membrane is characterized by being a nano-fat and platelet-rich fibrin composite membrane combined, and the nano-fat and platelet-rich fibrin composite membrane is prepared from autologous nano-fat, platelet-rich fibrin, quaternized chitosan and medical CaCl 2 The solution and the antioxidant are prepared by ion crosslinking;
the volume ratio of the formula raw materials of the nano-fat and platelet-rich fibrin composite membrane is as follows: the volume ratio of the platelet-rich fibrin to the autologous nano fat is 1: 5-1: 3, the volume ratio of the platelet-rich fibrin to the quaternized chitosan is 1: 5-1: 1, the volume ratio of the platelet-rich fibrin to the antioxidant is 1: 3-1: 1, and medical CaCl is added into each milliliter of the platelet-rich fibrin 2 The amount of the solution is 200-500. mu.L/mL.
2. The platelet-rich fibrin composite membrane according to claim 1, wherein the mass fraction of the quaternized chitosan is 2-6%.
3. The platelet-rich fibrin composite membrane according to claim 1, wherein the antioxidant is one or a mixture of two or more selected from gallic acid, tannic acid, theaflavin, dopamine, resveratrol, sodium phytate, and vitamin C.
4. The platelet-rich fibrin composite membrane according to claim 3, wherein the antioxidant is present in an amount of 1 to 4% by weight.
5. The preparation method of the platelet-rich fibrin composite membrane according to any one of claims 1 to 4, which comprises the following steps:
s1, mixing the autologous nano fat and the platelet-rich fibrin uniformly according to the volume ratio of the raw materials of the formula, and placing the mixture at 4 ℃ for later use;
s2, quaternizing the chitosan, the antioxidant and the medical CaCl under the normal temperature condition 2 The solution is stirred in proportion to form uniform mixed solution;
s3, stirring the mixture obtained in the step S1 and the mixed solution obtained in the step S2 uniformly at 4 ℃, transferring the mixture to a culture dish, and standing to form a membrane.
6. The method for preparing the platelet-rich fibrin composite membrane according to claim 5, wherein the method for preparing autologous nano-fat in step S1 is as follows:
(1) performing local swelling anesthesia around the navel of the abdomen of a patient, connecting a 20mL syringe with a liposuction needle with the aperture of 1mm, and performing radial suction under the condition of artificial negative pressure to obtain about 100mL of tiny granular fat;
(2) standing the injector for 10-15min, obviously layering the tiny fat particles and the swelling solution, removing the swelling solution, and washing the tiny fat particles with normal saline for 2-3 times;
(3) distributing the tiny particle fat in the step (2) into a 20mL injector, centrifuging for 3min at the rotating speed of 2000-3000rpm/min, removing water, and collecting the tiny particle fat;
(4) subpackaging the micro-particle fat collected in the step (3) in a 20mL syringe, connecting with a nano fat converter, continuously and mechanically cutting for 10-20 times, mechanically emulsifying, and continuously oscillating the emulsified fat for 1-2min under the condition of artificial negative pressure;
(5) centrifuging at the rotating speed of 2000-;
(6) filtering the emulsified fat with a filter screen with pore diameter of 0.5mm for 2-3 times, and removing the residual connective tissue to obtain fat emulsion, namely the autologous nano fat.
7. The method of claim 6, wherein the platelet-rich fibrin composite membrane is prepared in step S1 by the following steps:
(1) collecting 100mL of venous blood of a patient, and putting the collected blood into a centrifuge tube without anticoagulant;
(2) immediately centrifuging at the rotating speed of 1000-1600 rpm/min for 10-20min, and taking the middle gel layer to obtain platelet-rich fibrin;
(3) and storing the obtained platelet-rich fibrin at 4 ℃.
8. The application of the platelet-rich fibrin composite membrane according to any one of claims 1 to 7, in particular to the application of the platelet-rich fibrin composite membrane in diabetic foot wounds, dental tissue regeneration membranes and artificial skin.
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