Preparation method of platelet-rich plasma loaded electrostatic spinning membrane material

Abstract

The invention discloses a preparation method of an electrostatic spinning membrane material loaded with platelet-rich plasma, which is characterized by comprising the following steps: preparing an electrostatic nano spinning membrane; carrying out electrostatic nano spinning membrane treatment; the method comprises the following steps: extracting autologous venous blood of a patient under a sterile condition, collecting the blood into a centrifugal tube containing an anticoagulant, and enabling the platelet concentration to be greater than or equal to 1 * 10 < 9 > platelets / mu L through a secondary centrifugation method; in a culture dish, the electrostatic spinning membrane is soaked in the final PRP solution (the volume ratio is 1: 5-1: 10), and the soaking time is 24 h; the PRP-loaded electrostatic spinning membrane is used for isolating the wound surface of the uterine cavity. The PRP is stably loaded on the electrostatic spinning membrane, the defect that the liquid PRP is easy to lose is overcome, the bioactivity for promoting endometrial regeneration is added to the electrostatic spinning membrane which purely plays a supporting role, the electrostatic spinning membrane plays roles in supporting the uterine cavity and physically isolating to prevent recurrence of intrauterine adhesion, and meanwhile, PRP growth factors are continuously released to promote regeneration and repair of the endometrial.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a method for preparing an electrospun membrane material loaded with platelet-rich plasma. Background Technology

[0002] Intrauterine adhesions are common after intrauterine surgeries (such as induced abortion and curettage), with an incidence rate as high as 25%-30%. They are mainly caused by damage to the basal layer of the endometrium, which leads to the proliferation of fibrous connective tissue that replaces the normal endometrium, causing uterine cavity closure, reduced menstrual flow or even amenorrhea, which in turn leads to infertility or recurrent miscarriage.

[0003] Hysteroscopic adhesiolysis (TCRA) is currently the mainstream treatment, but due to the lack of an effective barrier to isolate the wound and the lack of regenerative active factors, the re-adhesion rate after surgery is still as high as 60%, and the pregnancy success rate is very low. Current research has invented an electrospun membrane that is placed in the uterine cavity after hysteroscopic surgery to provide a physical barrier to prevent intrauterine adhesions. However, the electrospun membrane alone lacks biological activity and cannot regulate the regenerative processes such as endometrial cell proliferation and angiogenesis, so it cannot actively promote endometrial repair.

[0004] Therefore, there is an urgent need for an implantable material that combines physical isolation with sustained bioactivity to simultaneously prevent adhesion recurrence and promote endometrial regeneration. PRP contains multiple growth factors that have been proven to effectively promote endometrial regeneration. If PRP can be loaded onto an electrospun membrane, the electrospun fiber network can physically block wound contact and inhibit fibrin deposition. At the same time, the PRP growth factors promote endometrial stromal cell proliferation, angiogenesis, and glandular remodeling, reversing fibrosis. This would enhance the therapeutic effect of electrospun membranes alone on intrauterine adhesions and effectively promote endometrial regeneration and repair. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing an electrospun membrane material loaded with platelet-rich plasma for preventing recurrence of adhesions and promoting endometrial regeneration.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: a method for preparing an electrospun membrane material loaded with platelet-rich plasma, characterized in that the preparation method includes the following steps: Preparation of electrostatic nanospun membrane: The spinning solution is drawn into a sterile syringe, the syringe is fixed on the injection pump, the injection rate is set, the high voltage power supply is connected to the needle, the injection pump and the high voltage power supply are started to spin the membrane, and the receiver is rotated or translated at a certain speed to collect the spun membrane. Electrostatic nano-spun membrane treatment: The spun membrane is sterilized by ethylene oxide gas. The sterilized spun membrane is transferred to a vacuum drying oven to allow any residue to evaporate completely. The dried spun membrane is then cut into the required shape and size. PRP preparation: Under aseptic conditions, autologous venous blood is drawn from the patient and collected into centrifuge tubes containing anticoagulant. The platelet concentration is ≥1×10⁹ platelets / μL by a two-stage centrifugation method. PRP activation: After centrifugation, add a sterile calcium ion source, centrifuge the activated PRP clot under sterile conditions, collect the supernatant, filter the supernatant through a 0.22μm sterile filter to remove clot fragments, and obtain the final PRP solution. Electrospun membrane and PRP immersion loading: In a petri dish, the electrospun membrane was immersed in the final PRP solution (volume ratio 1:5-1:10) for 24 hours; The electrospun membrane loaded with PRP is used to isolate the uterine cavity wound.

[0007] Preferably, the specific method for preparing the PRP is as follows: Under aseptic conditions, 20-60 ml of autologous venous blood is drawn from the patient and collected into a centrifuge tube containing 3.2%-3.8% sodium citrate for a second centrifugation. The tube is inverted and mixed several times to prevent clotting. The first centrifugation was performed at 1500 rpm for 20 minutes.

[0008] Separate the supernatant: Aspirate the upper plasma layer and a small amount of the red blood cell junction layer from the centrifuge tube and transfer them to a new sterile centrifuge tube. Avoid aspirating the red blood cells at the bottom.

[0009] Second centrifugation: Centrifuge the collected supernatant at 3500 rpm for 10 minutes.

[0010] Separating PPP from PRP concentrate: After centrifugation, platelet clumps form at the bottom of the tube, with anemic platelet plasma (PPP) on top. Carefully aspirate most of the PPP, leaving approximately 0.5-1 ml of PPP covering the platelet clumps.

[0011] Preferably, the specific method for PRP activation is as follows: Gently tap the tube wall with your finger or gently blow with a sterile pipette tip to resuspend the platelet clumps evenly in the remaining small amount of PPP, thus obtaining concentrated PRP; Take a small amount of PRP for platelet counting and calculate its concentration factor relative to whole blood; Add a sterile calcium ion source, such as a 10% CaCl2 solution or a thrombin-CaCl2 mixture, at a volume of 1 / 10 of the PRP volume, and mix gently. After activation, PRP forms a gel. Wait 0.3-2 hours to allow platelets to fully degranulate and release growth factors into the surrounding fluid. The activated PRP clots were centrifuged under aseptic conditions at a centrifugal force of 1500-2000g for 5-10 minutes. Collect the supernatant and filter it through a 0.22 μm sterile filter to remove any possible tiny clots or fragments.

[0012] Preferably, the specific method for impregnating and loading the electrospun membrane with PRP is as follows: Inside the sterile operating table, place the prepared sterile electrospun membrane into a sterile petri dish or multi-well plate; Carefully add a sufficient amount of freshly prepared PRP release solution to the membrane surface, ensuring that the membrane is completely submerged; Cover the petri dish / plate and place it in a 4°C refrigerator or at room temperature for 24 hours to soak. After soaking, aspirate the soaking solution and quickly rinse the membrane surface 1-2 times with a small amount of sterile PBS or basal culture medium to remove unbound or loosely adsorbed proteins / growth factors on the membrane surface.

[0013] Preferably, it also includes methods for short-term preservation and long-term storage; The short-term preservation method is as follows: transfer the electrospun membrane loaded with PRP growth factor, place it at 4°C, and immerse it in a small amount of sterile PBS or basal culture medium for short-term preservation. The long-term storage method is as follows: freeze-dry the electrospun membrane loaded with PRP growth factor, and then store it in a sealed, light-proof low-temperature environment at -20°C to -80°C. It needs to be rehydrated under sterile conditions before use.

[0014] Preferably, the spun film is cut into 1.5×2cm sheets.

[0015] An electrospun membrane material loaded with platelet-rich plasma is prepared by any of the above-described preparation methods.

[0016] Compared with the prior art, the advantages of the present invention are as follows: This invention features a novel composite design: PRP is stably loaded onto an electrospun membrane, overcoming the defect of easy loss of liquid PRP, and adding bioactivity that promotes endometrial regeneration to the electrospun membrane, which only serves a supporting function.

[0017] Synergistic effect: The electrospun membrane supports the uterine cavity and acts as a physical barrier to prevent recurrence of intrauterine adhesions. At the same time, PRP growth factors are continuously released to promote endometrial regeneration and repair. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a flowchart of a method for preparing an electrospun membrane material loaded with platelet-rich plasma according to the present invention; Figure 2 This is a comparison chart showing the release of growth factors between the electrospun membrane with added PRP and the electrospun membrane without PRP loading according to the present invention. Figure 3 This is a comparison of the results of repairing rat endometrium with electrospun membrane loaded with platelet-rich plasma (PRP) and without PRP loading. Detailed Implementation

[0020] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby providing a clearer and more explicit definition of the scope of protection of the present invention.

[0021] like Figure 1 As shown, the present invention provides a method for preparing an electrospun membrane material loaded with platelet-rich plasma, characterized in that the preparation method includes the following steps: Preparation of electrostatic nanospun membrane: The spinning solution is drawn into a sterile syringe, the syringe is fixed on the injection pump, the injection rate is set, the high voltage power supply is connected to the needle, the injection pump and the high voltage power supply are started to spin the membrane, and the receiver is rotated or translated at a certain speed to collect the spun membrane. Electrostatic nano-spun membrane treatment: The spun membrane is sterilized by ethylene oxide gas. The sterilized spun membrane is transferred to a vacuum drying oven to allow any residue to evaporate completely. The dried spun membrane is then cut into the required shape and size. The preferred sterilization method is ethylene oxide gas sterilization (suitable for most polymers, but requires thorough desorption). Second choice: Ultraviolet irradiation sterilization (surface sterilization, short time).

[0022] The aforementioned electrospun nanofiber membranes can also be purchased directly, specifically from the electrospun nanofiber membranes provided by Nomaier Company.

[0023] PRP preparation: Under aseptic conditions, autologous venous blood is drawn from the patient and collected into centrifuge tubes containing anticoagulant. The platelet concentration is ≥1×10⁹ platelets / μL by a two-stage centrifugation method. PRP activation: After centrifugation, add a sterile calcium ion source, centrifuge the activated PRP clot under sterile conditions, collect the supernatant, filter the supernatant through a 0.22μm sterile filter to remove clot fragments, and obtain the final PRP solution. Electrospun membrane and PRP immersion loading: In a petri dish, the electrospun membrane was immersed in the final PRP solution (volume ratio 1:5-1:10) for 24 hours; The electrospun membrane loaded with PRP is used to isolate the uterine cavity wound.

[0024] In this embodiment, the specific method for preparing PRP is as follows: Under aseptic conditions, 20-60 ml of autologous venous blood is drawn from the patient and collected into a centrifuge tube containing 3.2%-3.8% sodium citrate for a second centrifugation. The tube is inverted and mixed several times to prevent clotting. The first centrifugation was performed at 1500 rpm for 20 minutes.

[0025] Separate the supernatant: Aspirate the upper plasma layer and a small amount of the red blood cell junction layer from the centrifuge tube and transfer them to a new sterile centrifuge tube. Avoid aspirating the red blood cells at the bottom.

[0026] Second centrifugation: Centrifuge the collected supernatant at 3500 rpm for 10 minutes.

[0027] Separating PPP from PRP concentrate: After centrifugation, platelet clumps form at the bottom of the tube, with anemic platelet plasma (PPP) on top. Carefully aspirate most of the PPP, leaving approximately 0.5-1 ml of PPP covering the platelet clumps.

[0028] In this embodiment, the specific method for PRP activation is as follows: Gently tap the tube wall with your finger or gently blow with a sterile pipette tip to resuspend the platelet clumps evenly in the remaining small amount of PPP, thus obtaining concentrated PRP; Take a small amount of PRP for platelet counting and calculate its concentration factor relative to whole blood; Add a sterile calcium ion source, such as a 10% CaCl2 solution or a thrombin-CaCl2 mixture, at a volume of 1 / 10 of the PRP volume, and mix gently. After activation, PRP forms a gel-like substance, which is a fibrin clot. Waiting 0.3-2 hours allows the platelets to fully degranulate, releasing growth factors (such as PDGF, TGF-β, VEGF, EGF, IGF, etc.) into the surrounding fluid. A comparison of growth factor release between electrospun membranes with added PRP and those without PRP loading, such as... Figure 2 As shown; The activated PRP clots were centrifuged under aseptic conditions at a centrifugal force of 1500-2000g for 5-10 minutes. Collect the supernatant and filter it through a 0.22 μm sterile filter to remove any possible tiny clots or fragments.

[0029] In this embodiment, the specific method for impregnating and loading the electrospun membrane with PRP is as follows: Inside the sterile operating table, place the prepared sterile electrospun membrane into a sterile petri dish or multi-well plate; Carefully add a sufficient amount of freshly prepared PRP release solution to the membrane surface, ensuring that the membrane is completely submerged; Cover the petri dish / plate and place it in a 4°C refrigerator or at room temperature for 24 hours to soak. After soaking, the soaking solution is aspirated, and the membrane surface is quickly rinsed 1-2 times with a small amount of sterile PBS or basal culture medium to remove unbound or loosely adsorbed proteins / growth factors on the membrane surface. The purpose is to reduce the negative effects of possible inhibitors or excessively high concentrations.

[0030] This embodiment also includes short-term preservation and long-term storage methods; The short-term preservation method is as follows: transfer the electrospun membrane loaded with PRP growth factor, place it at 4°C, and immerse it in a small amount of sterile PBS or basal culture medium for short-term preservation. The long-term storage method is as follows: freeze-dry the electrospun membrane loaded with PRP growth factor, and then store it in a sealed, light-proof low-temperature environment at -20°C to -80°C. It needs to be rehydrated under sterile conditions before use.

[0031] In this embodiment, the spun film is cut into 1.5×2cm sheets.

[0032] An electrospun membrane material loaded with platelet-rich plasma is prepared by any of the above-described preparation methods.

[0033] Electrospun membranes loaded with platelet-rich plasma were used to repair rat endometrium, while ordinary electrospun membranes were also used. After a certain period, monitoring was performed, and it was found that the platelet-rich plasma-loaded electrospun membrane promoted endometrial regeneration and repair. Figure 3 As shown.

[0034] Although embodiments of the present invention have been described in conjunction with the accompanying drawings, the patent owner may make various modifications or alterations within the scope of the appended claims, as long as they do not exceed the protection scope described in the claims of the present invention, they shall be within the protection scope of the present invention.

Claims

1. A method for preparing an electrospun membrane material loaded with platelet-rich plasma, characterized in that, The preparation method includes the following steps: Preparation of electrostatic nanospun membrane: The spinning solution is drawn into a sterile syringe, the syringe is fixed on the injection pump, the injection rate is set, the high voltage power supply is connected to the needle, the injection pump and the high voltage power supply are started to spin the membrane, and the receiver is rotated or translated at a certain speed to collect the spun membrane. Electrostatic nano-spun membrane treatment: The spun membrane is sterilized by ethylene oxide gas. The sterilized spun membrane is transferred to a vacuum drying oven to allow any residue to evaporate completely. The dried spun membrane is then cut into the required shape and size. PRP preparation: Under aseptic conditions, autologous venous blood is drawn from the patient and collected into centrifuge tubes containing anticoagulant. The blood is then centrifuged twice to achieve a platelet concentration ≥1×10⁻⁶. 9 platelets / μL; PRP activation: After centrifugation, add a sterile calcium ion source, centrifuge the activated PRP clot under sterile conditions, collect the supernatant, filter the supernatant through a 0.22μm sterile filter to remove clot fragments, and obtain the final PRP solution. Electrospun membrane and PRP immersion loading: In a petri dish, the electrospun membrane was immersed in the final PRP solution (volume ratio 1:5-1:10) for 24 hours; The electrospun membrane loaded with PRP is used to isolate the uterine cavity wound.

2. The method for preparing an electrospun membrane material loaded with platelet-rich plasma according to claim 2, characterized in that, The specific method for preparing the PRP is as follows: Under aseptic conditions, 20-60 ml of autologous venous blood is drawn from the patient and collected into a centrifuge tube containing 3.2%-3.8% sodium citrate for a second centrifugation. The tube is inverted and mixed several times to prevent clotting. The first one Centrifugation: Centrifuge at 1500 rpm for 20 minutes; Separate the supernatant: Aspirate the upper plasma layer and a small amount of the red blood cell junction layer from the centrifuge tube and transfer them to a new sterile centrifuge tube. Avoid aspirating the red blood cells at the bottom; Second centrifugation: Centrifuge the collected supernatant at 3500 rpm for 10 minutes; Separating PPP from PRP concentrate: After centrifugation, platelet clumps form at the bottom of the tube, with anemic platelet plasma (PPP) on top. Carefully aspirate most of the PPP, leaving approximately 0.5-1 ml of PPP covering the platelet clumps.

3. The method for preparing an electrospun membrane material loaded with platelet-rich plasma according to claim 1, characterized in that, The specific method for PRP activation is as follows: Gently tap the tube wall with your finger or gently blow with a sterile pipette tip to resuspend the platelet clumps evenly in the remaining small amount of PPP, thus obtaining concentrated PRP; Take a small amount of PRP for platelet counting and calculate its concentration factor relative to whole blood; Add a sterile calcium ion source, such as a 10% CaCl2 solution or a thrombin-CaCl2 mixture, at a volume of 1 / 10 of the PRP volume, and mix gently. After activation, PRP forms a gel. Wait 0.3-2 hours to allow platelets to fully degranulate and release growth factors into the surrounding fluid. The activated PRP clots were centrifuged under aseptic conditions at a centrifugal force of 1500-2000g for 5-10 minutes. Collect the supernatant and filter it through a 0.22 μm sterile filter to remove any possible tiny clots or fragments.

4. The method for preparing an electrospun membrane material loaded with platelet-rich plasma according to claim 1, characterized in that, The specific method for impregnating and loading the electrospun membrane with PRP is as follows: Inside the sterile operating table, place the prepared sterile electrospun membrane into a sterile petri dish or multi-well plate; Carefully add a sufficient amount of freshly prepared PRP release solution to the membrane surface to ensure that the membrane is completely submerged; Cover the petri dish / plate and place it in a 4°C refrigerator or at room temperature for 24 hours to soak. After soaking, aspirate the soaking solution and quickly rinse the membrane surface 1-2 times with a small amount of sterile PBS or basal culture medium to remove unbound or loosely adsorbed proteins / growth factors on the membrane surface.

5. The method for preparing an electrospun membrane material loaded with platelet-rich plasma according to claim 1, characterized in that, It also includes methods for short-term preservation and long-term storage; The short-term preservation method is as follows: transfer the electrospun membrane loaded with PRP growth factor, place it at 4°C, and immerse it in a small amount of sterile PBS or basal culture medium for short-term preservation. The long-term storage method is as follows: freeze-dry the electrospun membrane loaded with PRP growth factor, and then store it in a sealed, light-proof low-temperature environment at -20°C to -80°C. It needs to be rehydrated under sterile conditions before use.

6. The method for preparing an electrospun membrane material loaded with platelet-rich plasma according to claim 1, characterized in that: The spun film is cut into 1.5×2cm sheets.

7. An electrospun membrane material loaded with platelet-rich plasma, characterized in that: It is prepared by any one of the preparation methods described in claims 1-6.

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