Platelet-rich plasma for anti-aging and promoting tissue repair and regeneration, preparation method and application thereof

Abstract

The present invention relates to the field of biomedicine and discloses a platelet-rich plasma for anti-aging and promoting tissue repair and regeneration, a preparation method, and an application thereof, comprising the following steps: obtaining concentrated platelet plasma, and treating the concentrated platelet plasma with low-intensity pulsed ultrasound; the treatment conditions are one of the following treatment methods: 1) the treatment power is 2 to 5 W / cm 2 , the pulse period is 50%, and the ultrasonic treatment time is 2~10 min; 2) First, the first ultrasonic treatment is performed: using 0.2~1.0 W / cm 2 , the pulse period is 50%, ultrasonic treatment is 1~5 min; then the second ultrasonic treatment is carried out: 1.5~5.0 W / cm 2 , the pulse period is 50%, and the ultrasonic treatment is 1 to 5 minutes; the present invention adopts a special ultrasonic treatment system to obtain a PRR product with special activation and lysis characteristics. The product has active substances that are beneficial to tissue repair and regeneration and anti-aging, and shows excellent effects in promoting cell proliferation, cell migration and wound healing.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to platelet-rich plasma for resisting aging and promoting tissue repair and regeneration, a preparation method and application thereof. Background Art

[0002] Platelets play a vital role in tissue repair and wound healing. Platelet-rich plasma (PRP) is a concentrated platelet-rich plasma product. Typically, doctors draw blood from patients and then centrifuge it to produce PRP, which contains 3-6 times more platelets than normal blood. When activated, platelets release a large number of bioactive factors that promote tissue repair and growth, making it widely used in surgical treatments. However, the rapid release of these bioactive factors after activation and their susceptibility to degradation by enzymes on the wound surface significantly limit the clinical application of PRP. Platelet lysate, a purified extract from platelet-rich plasma, contains a wide range of growth factors, cytokines, and proteins essential for cell proliferation. Human platelet lysate is a highly effective cell culture supplement that effectively supports the growth of human cells, including mesenchymal stem cells. Platelet lysate is often used in place of various serums, such as fetal bovine serum and human AB serum, in both scientific research and clinical practice. Platelet activation and lysis are crucial for their therapeutic efficacy, and spatiotemporally controlled activation and lysis may enhance the efficacy of any treatment involving PRP.

[0003] Upon activation, platelets release a variety of bioactive substances, including cytokines, growth factors, inflammatory mediators, and metabolites. These substances play important physiological roles in hemostasis, tissue repair, inflammation, and immune responses. For ex vivo platelet activation, physicians can trigger platelet activation in PRP by adding a typical activator, such as thrombin, and then administering the activated PRP to the site. For such ex vivo applications, bovine thrombin can be used to induce platelet activation. However, the use of animal-based thrombin or its alternatives is often expensive and can cause allergic reactions. Platelet activation in PRP can be triggered by varying concentrations of calcium ions (calcium chloride / calcium gluconate). However, this activation method results in rapid and difficult-to-control release of cytokines from platelets, resulting in a short-lived and unsustainable biological effect. The rapidly formed fibrin network has poor elasticity and stability and is prone to contraction, which hinders the attraction and engraftment of stem / progenitor cells in vivo. Activated PRP also poorly adheres to commonly used surgical dressings, leading to the loss of most of its active ingredients. Exposing PRP samples to various types of light (e.g., infrared) can trigger platelet activation and growth factor release without subsequent coagulation. However, this process is time-consuming and the sample's light exposure time can be long, which will increase the total treatment time. In addition, due to the limited tissue penetration of light, this method is not conducive to in situ activation in vivo in deep tissue applications. These shortcomings limit the efficiency and safety of PRP application in clinical practice and need to be overcome by improving preparation methods and clinical application technologies.

[0004] Platelet lysis methods involve techniques such as sonication and freeze-thaw cycles. Ultrasonic homogenization is a commonly used laboratory procedure for cell lysis. Sound waves with frequencies exceeding 20 kHz rapidly compress and decompress dissolved gases, causing violent collisions that rupture cell membranes and subsequently release intracellular proteins. The ultrasonic waterbath method uses a sealed container to place the PRP sample in a water bath, through which ultrasound waves are transmitted. Another method involves inserting an ultrasound probe into the PRP sample to transmit ultrasound directly, providing more efficient energy transmission but requiring specialized equipment such as sterile ultrasound cannulas. However, this high-power ultrasound exposure for 5–30 minutes can inactivate some active substances through strong mechanical action, cavitation, and heat generation. Freeze-thaw cycles are another commonly used platelet lysis method. In this process, PRP is first frozen at -80°C or in liquid nitrogen until completely solidified and then completely thawed in a 37°C water bath. This process is repeated five times, followed by centrifugation at 10,000 × g for 10 minutes, and the supernatant is aspirated. The supernatant contains platelet lysate, which can be used for various experimental or therapeutic purposes. However, this method is time-consuming, requires deep-freezing equipment, and is a cumbersome process. Summary of the Invention

[0005] In view of the problems existing in the prior art, the present invention provides a platelet-rich plasma for resisting aging and promoting tissue repair and regeneration, a preparation method and application thereof.

[0006] The technical solution adopted in the present invention is:

[0007] A method for preparing platelet-rich plasma for anti-aging and promoting tissue repair and regeneration comprises the following steps:

[0008] obtaining platelet-concentrated plasma and treating the platelet-concentrated plasma with low-intensity pulsed ultrasound;

[0009] The processing condition is one of the following processing methods:

[0010] 1) Processing power is 2~5 W / cm 2 , the pulse period is 50%, and the ultrasonic treatment time is 2~10 min;

[0011] 2) First, perform the first ultrasonic treatment: use 0.2~1.0 W / cm 2 , pulse period is 50%, ultrasonic treatment is 1~5min;

[0012] Then the second ultrasonic treatment was carried out: 1.5~5.0 W / cm 2 , pulse period is 50%, ultrasonic treatment is 1~5min;

[0013] The desired platelet-rich plasma can be obtained after ultrasonic treatment.

[0014] Furthermore, an ultrasonic therapeutic apparatus is used for ultrasonic treatment.

[0015] Furthermore, the platelet concentrated plasma preparation process is as follows:

[0016] Anticoagulant is added to the blood and concentrated platelet plasma is obtained after centrifugation; the anticoagulant is sodium citrate anticoagulant, and the added volume accounts for 1% of the blood volume.

[0017] Furthermore, the concentrated platelet plasma is placed in a well culture plate, the probe of the ultrasonic therapeutic device is located at the bottom of the well plate, and the center of the probe is facing the center of the well; a 0.5-1 cm gel coupling agent is provided between the probe and the well plate.

[0018] Furthermore, the ultrasonic frequency in the ultrasonic treatment is 1 MHz to 3 MHz.

[0019] Furthermore, the plasma after the first ultrasonic treatment is allowed to stand at 37° C. in air containing 5% CO 2 for 0.5 to 1 h.

[0020] A platelet-rich plasma that fights aging and promotes tissue repair and regeneration.

[0021] The invention discloses an application of platelet-rich plasma for resisting aging and promoting tissue repair and regeneration, wherein the plasma is used for preparing medicines.

[0022] Furthermore, the drug is one of anti-inflammatory drugs, scald drugs, trauma drugs, and anti-aging drugs.

[0023] The beneficial effects of the present invention are:

[0024] The present invention adopts a special ultrasonic treatment system to obtain a PRR product with special activation and lysis characteristics. It contains active substances (growth factors, metabolites, etc.) that are beneficial to tissue repair and regeneration and anti-aging, and shows excellent effects in promoting cell proliferation, cell migration and wound healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a graph of plasma after the first ultrasonic treatment in Comparative Example 1 of the present invention.

[0026] Figure 2 This is a graph of plasma after the first ultrasonic treatment in Example 1 of the present invention.

[0027] Figure 3 Schematic diagram comparing the content of platelet factor IV PF4 in PRP obtained in Example 1 and Example 2 of the present invention.

[0028] Figure 4 These are scanning electron microscope (SEM) images of the PRP products obtained in Example 1, Comparative Example 2, and Comparative Example 3 of the present invention.

[0029] Figure 5 Schematic diagram comparing the platelet aggregation test results of the PRP products obtained in Example 1, Comparative Example 2 and Comparative Example 3 of the present invention.

[0030] Figure 6 Schematic diagram comparing the platelet aggregation ability of the PRP products obtained in Examples 3, 5, 8, and Comparative Examples 7 to 10 of the present invention and the untreated PRP products; and schematic diagram comparing the flow cytometry assay for the activation marker CD62P.

[0031] Figure 7 Schematic diagram of flow cytometry experiments comparing the platelet activation marker CD62P in the PRP products obtained in Examples 3, 5, 8, and Comparative Examples 7 to 10 of the present invention and in untreated PRP products.

[0032] Figure 8 This is a schematic diagram comparing the growth factor contents in the PRP products obtained in Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, and Example 3 of the present invention, where a represents the PF4 content, b represents the FGF content, and c represents the TGFβ content.

[0033] Figure 9 This is a schematic diagram comparing the platelet metabolites in the PRP products obtained in Example 1, Comparative Example 2, and Comparative Example 3 of the present invention, where a is α-ketoglutaric acid, b is taurine, c is 12-hydroxyeicosatrienoic acid, and d is spermidine.

[0034] Figure 10 Schematic diagram comparing the cell proliferation of the PRP products obtained in Examples 3, 5, 8, and Comparative Examples 7 to 10 of the present invention and the untreated PRP products after co-culturing with human keratinocytes Hacat.

[0035] Figure 11 This is a schematic diagram comparing the cell proliferation after co-culture of the PRP products obtained in Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 5, and Comparative Example 6 with human keratinocytes Hacat, wherein a is the result of Example 1, Comparative Example 2, Comparative Example 3, and untreated PRP products in promoting the proliferation of human keratinocytes Hacat, and b is the result of Example 1, Comparative Example 5, Comparative Example 6, and untreated PRP products in promoting the proliferation of human keratinocytes Hacat.

[0036] Figure 12 Schematic diagram comparing the cell proliferation after co-culture of Example 1, Example 3, and untreated PRP product with human keratinocytes Hacat.

[0037] Figure 13 This is a schematic diagram comparing the proliferation of PRP products obtained in Example 1, Comparative Example 2, and Comparative Example 3 of the present invention after co-culture with mouse primary fibroblasts.

[0038] Figure 14 Schematic diagram comparing the migration ability of PRP products obtained in Example 1, Comparative Example 2, and Comparative Example 3 of the present invention after co-culture with mouse primary fibroblasts.

[0039] Figure 15 This is a comparison chart of the therapeutic effects of the PRP products obtained from Example 1, Comparative Example 2, and Comparative Example 3 of the present invention on deep second-degree burn wounds in rats. DETAILED DESCRIPTION

[0040] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0041] A method for preparing platelet-rich plasma for anti-aging and promoting tissue repair and regeneration comprises the following steps:

[0042] Obtaining Platelet Concentrated Plasma: The platelet concentrated plasma preparation process is as follows:

[0043] Anticoagulant is added to the blood and concentrated platelet plasma is obtained after centrifugation; the anticoagulant is sodium citrate anticoagulant, and the added volume accounts for 1% of the blood volume.

[0044] Low-intensity pulsed ultrasound is used to treat platelet-concentrated plasma (PCP). Ultrasonic treatment is performed using an ultrasonic therapy device. The PCP is placed in a well culture plate. The ultrasonic therapy device probe is located at the bottom of the plate, with the probe center facing the center of the well. A 0.5-1 cm layer of gel coupling agent is placed between the probe and the plate. The presence of gel coupling agent significantly impacts the final product's performance. Ultrasonic heat generation disrupts platelet function. If the gel coupling agent is less than 0.5 cm, excessive ultrasonic heat transfer to the PRP can lead to overactivation of the platelets and irreversible aggregation, compromising subsequent PRP use.

[0045] The processing condition is one of the following processing methods:

[0046] 1) Processing power is 2~5 W / cm 2 , the pulse period is 50%, the ultrasonic treatment time is 2~10 min; the ultrasonic frequency is 1 MHz.

[0047] 2) First, perform the first ultrasonic treatment: use 0.2~1.0 W / cm 2 , pulse period is 50%, ultrasonic treatment is 1~5min; ultrasonic frequency is 1 MHz.

[0048] Plasma is incubated at 37°C in an atmosphere of 5% CO2 for 0.5 to 1 hour. This incubation process is crucial for the production and secretion of active substances by platelets after subsequent ultrasonic treatment.

[0049] Then the second ultrasonic treatment was carried out: 1.5~5.0 W / cm 2 , pulse period is 50%, ultrasonic treatment time is 1~5min; ultrasonic frequency is 1~3 MHz.

[0050] The desired platelet-rich plasma can be obtained after ultrasonic treatment.

[0051] Example 1

[0052] Collect whole blood into an anticoagulant tube, add 1% of the whole blood volume of the anticoagulant sodium citrate, and centrifuge at room temperature for 120*15 minutes to obtain the required platelet concentrated plasma.

[0053] Place the platelet concentrate in a sterile, enzyme-free well culture plate. The number of wells should be adjusted as needed, with each well containing approximately 1 / 2 of the total well volume. The procedure must be performed under sterile conditions, and all consumables must be sterile and enzyme-free.

[0054] First, perform the first ultrasonic treatment:

[0055] Ultrasonic therapy device, 2 cm 2 / 5 cm 2probe, ultrasonic power is 0.8 W / cm 2 The ultrasonic treatment was performed at a 50% pulse period and a probe frequency of 1 MHz for 3 minutes. During ultrasonic treatment, the probe should be positioned at the bottom of the plate, with the center of the probe facing the center of each well. A 0.8 cm layer of gel couplant was placed between the probe and the plate.

[0056] After the first ultrasonic treatment, the well plate was placed in a cell culture incubator at 37°C with 95% air and 5% CO2 for 1 hour.

[0057] Then a second sonication treatment was performed:

[0058] Ultrasonic therapy device, 2 cm 2 / 5 cm 2 probe, ultrasonic power is 2 W / cm 2 The ultrasonic treatment was performed at a 50% pulse period and a probe frequency of 3 MHz for 3 minutes. During ultrasonic treatment, the probe should be positioned at the bottom of the plate, with the center of the probe facing the center of each well. A 0.8 cm layer of gel couplant was placed between the probe and the plate.

[0059] After ultrasonic treatment, the obtained PRP products (PRP (US) and PRP (US) 0.8 + 2W in the figure) can be used directly or stored at -80 °C and used as needed.

[0060] Example 2

[0061] Collect whole blood into an anticoagulant tube, add 1% of the whole blood volume of the anticoagulant sodium citrate, and centrifuge at room temperature for 120*15 minutes to obtain the required platelet concentrated plasma.

[0062] Place the platelet concentrate in a sterile, enzyme-free well culture plate. The number of wells should be adjusted as needed, with each well containing approximately 1 / 2 of the total well volume. The procedure must be performed under sterile conditions, and all consumables must be sterile and enzyme-free.

[0063] First, perform the first ultrasonic treatment:

[0064] Ultrasonic therapy device, 2 cm 2 / 5 cm 2 probe, ultrasonic power is 0.8 W / cm 2 The ultrasonic treatment was performed at a 50% pulse period and a probe frequency of 1 MHz for 3 minutes. During ultrasonic treatment, the probe should be positioned at the bottom of the plate, with the center of the probe facing the center of each well. A 0.8 cm layer of gel couplant was placed between the probe and the plate.

[0065] Then a second sonication treatment was performed:

[0066] Ultrasonic therapy device, 2 cm 2 / 5 cm 2 probe, ultrasonic power is 2 W / cm 2 The ultrasonic treatment was performed at a 50% pulse period and a probe frequency of 3 MHz for 3 minutes. During ultrasonic treatment, the probe should be positioned at the bottom of the plate, with the center of the probe facing the center of each well. A 0.8 cm layer of gel couplant was placed between the probe and the plate.

[0067] After ultrasonic treatment, it can be used directly or stored at -80 °C and used as needed.

[0068] Example 3

[0069] Collect whole blood into an anticoagulant tube, add 1% of the whole blood volume of the anticoagulant sodium citrate, and centrifuge at room temperature for 120*15 minutes to obtain the required platelet concentrated plasma.

[0070] Place the platelet concentrate in a sterile, enzyme-free well culture plate. The number of wells should be adjusted as needed, with each well containing approximately 1 / 2 of the total well volume. The procedure must be performed under sterile conditions, and all consumables must be sterile and enzyme-free.

[0071] Ultrasonic therapy device, 2 cm 2 / 5 cm 2 probe, ultrasonic power is 2.0 W / cm 2 The ultrasonic treatment was performed with a pulse period of 50%, a probe frequency of 1 MHz, and 3 minutes of sonication. During sonication, the probe should be positioned at the bottom of the plate, with the center of the probe aligned with the center of each well. A 0.8 cm layer of gel coupling agent was placed between the probe and the plate. The resulting product is referred to as PRP(US)2W.

[0072] Example 4

[0073] Collect whole blood into an anticoagulant tube, add 1% of the whole blood volume of the anticoagulant sodium citrate, and centrifuge at room temperature for 120*15 minutes to obtain the required platelet concentrated plasma.

[0074] Place the platelet concentrate in a sterile, enzyme-free well culture plate. The number of wells should be adjusted as needed, with each well containing approximately 1 / 2 of the total well volume. The procedure must be performed under sterile conditions, and all consumables must be sterile and enzyme-free.

[0075] Ultrasonic therapy device, 2 cm 2 / 5 cm 2 probe, ultrasonic power 5.0 W / cm 2The ultrasonic treatment was performed at a 50% pulse period and a probe frequency of 1 MHz for 3 minutes. During ultrasonic treatment, the probe should be positioned at the bottom of the plate, with the center of the probe facing the center of each well. A 0.8 cm layer of gel couplant was placed between the probe and the plate.

[0076] Example 5

[0077] Collect whole blood into an anticoagulant tube, add 1% of the whole blood volume of the anticoagulant sodium citrate, and centrifuge at room temperature for 120*15 minutes to obtain the required platelet concentrated plasma.

[0078] Place the platelet concentrate in a sterile, enzyme-free well culture plate. The number of wells should be adjusted as needed, with each well containing approximately 1 / 2 of the total well volume. The procedure must be performed under sterile conditions, and all consumables must be sterile and enzyme-free.

[0079] Ultrasonic therapy device, 2 cm 2 / 5 cm 2 probe, ultrasonic power is 2.5 W / cm 2 The ultrasonic treatment was performed with a pulse period of 50%, a probe frequency of 1 MHz, and 3 minutes of sonication. During sonication, the probe should be positioned at the bottom of the plate, with the center of the probe aligned with the center of each well. A 0.8 cm layer of gel couplant was placed between the probe and the plate. The resulting product is referred to as PRP (US) 2.5W.

[0080] Example 6

[0081] Collect whole blood into an anticoagulant tube, add 1% of the whole blood volume of the anticoagulant sodium citrate, and centrifuge at room temperature for 120*15 minutes to obtain the required platelet concentrated plasma.

[0082] Place the platelet concentrate in a sterile, enzyme-free well culture plate. The number of wells should be adjusted as needed, with each well containing approximately 1 / 2 of the total well volume. The procedure must be performed under sterile conditions, and all consumables must be sterile and enzyme-free.

[0083] First, perform the first ultrasonic treatment:

[0084] Ultrasonic therapy device, 2 cm 2 / 5 cm 2 probe, ultrasonic power is 0.2 W / cm 2 The ultrasonic treatment was performed at a pulse period of 50%, a probe frequency of 3 MHz, and 5 minutes of sonication. During sonication, the probe should be positioned at the bottom of the plate, with the center of the probe facing the center of each well. A 0.5 cm layer of gel couplant was placed between the probe and the plate.

[0085] After the first ultrasonic treatment, the well plate was placed in a cell culture incubator at 37°C with 95% air and 5% CO2 for 0.5 hours.

[0086] Then a second sonication treatment was performed:

[0087] Ultrasonic therapy device, 2 cm 2 / 5 cm 2 probe, ultrasonic power 5.0 W / cm 2 The ultrasonic treatment was performed at a pulse period of 50%, a probe frequency of 1 MHz, and a duration of 1 minute. During ultrasonic treatment, the probe should be positioned at the bottom of the well plate, with the center of the probe facing the center of each well. A 1 cm layer of gel couplant was placed between the probe and the well plate.

[0088] After ultrasonic treatment, the obtained PRP product can be used directly or stored at -80 °C and used as needed.

[0089] Example 7

[0090] Collect whole blood into an anticoagulant tube, add 1% of the whole blood volume of the anticoagulant sodium citrate, and centrifuge at room temperature for 120*15 minutes to obtain the required platelet concentrated plasma.

[0091] Place the platelet concentrate in a sterile, enzyme-free well culture plate. The number of wells should be adjusted as needed, with each well containing approximately 1 / 2 of the total well volume. The procedure must be performed under sterile conditions, and all consumables must be sterile and enzyme-free.

[0092] First, perform the first ultrasonic treatment:

[0093] Ultrasonic therapy device, 2 cm 2 / 5 cm 2 probe, ultrasonic power is 1.0 W / cm 2 The ultrasonic treatment was performed at a 50% pulse period, a probe frequency of 1 MHz, and a duration of 1 minute. During ultrasonic treatment, the probe should be positioned at the bottom of the plate, with the center of the probe facing the center of each well. A 0.5 cm layer of gel couplant was placed between the probe and the plate.

[0094] After the first ultrasonic treatment, the well plate was placed in a cell culture incubator at 37°C with 95% air and 5% CO2 for 0.5 hours.

[0095] Then a second sonication treatment was performed:

[0096] Ultrasonic therapy device, 2 cm 2 / 5 cm 2 probe, ultrasonic power of 1.5 W / cm 2The ultrasonic treatment was performed at a pulse period of 50%, a probe frequency of 3 MHz, and 5 minutes of sonication. During sonication, the probe should be positioned at the bottom of the plate, with the center of the probe facing the center of each well. A 0.8 cm layer of gel couplant was placed between the probe and the plate.

[0097] After ultrasonic treatment, the obtained PRP product can be used directly or stored at -80 °C and used as needed.

[0098] Example 8

[0099] Collect whole blood into an anticoagulant tube, add 1% of the whole blood volume of the anticoagulant sodium citrate, and centrifuge at room temperature for 120*15 minutes to obtain the required platelet concentrated plasma.

[0100] Place the platelet concentrate in a sterile, enzyme-free well culture plate. The number of wells should be adjusted as needed, with each well containing approximately 1 / 2 of the total well volume. The procedure must be performed under sterile conditions, and all consumables must be sterile and enzyme-free.

[0101] Ultrasonic therapy device, 2 cm 2 / 5 cm 2 Probe, ultrasonic power is 3.0W / cm 2 The ultrasonic treatment was performed with a pulse period of 50%, a probe frequency of 1 MHz, and 3 minutes of sonication. During sonication, the probe should be positioned at the bottom of the plate, with the center of the probe aligned with the center of each well. A 0.8 cm layer of gel couplant was placed between the probe and the plate. The resulting product is referred to as PRP(US)3.0W.

[0102] Comparative Example 1

[0103] The other steps of this comparative example are the same as those of Example 1, except that a 0.2 cm gel coupling agent is provided between the probe and the orifice plate during the ultrasonic treatment.

[0104] Comparative Example 2

[0105] This comparative example is a PRP product obtained by processing with the existing repeated freeze-thaw method, that is, after PL treatment in the accompanying drawing, and PL in the accompanying drawing is the PRP product obtained in this comparative example.

[0106] Here’s how:

[0107] The whole blood was added to the anticoagulant tube, and the anticoagulant sodium citrate was added to the whole blood, the mass of which accounted for 1% of the whole blood mass. The whole blood was centrifuged at 120g for 15 minutes at room temperature to remove the red blood cells and prepare concentrated platelets.

[0108] Place the platelet at -80°C for 10 min and then incubate in a water bath at 37°C for 3 min, repeating this process three times to prepare the platelet-rich plasma by repeated freeze-thaw cycles.

[0109] Comparative Example 3

[0110] This comparative example is activated platelet-rich plasma obtained by treatment with thrombin + calcium method, referred to as PRP (AA). This abbreviation in the accompanying drawings refers to the PRP product obtained in comparative example 3.

[0111] The whole blood was added to the anticoagulant tube, and the anticoagulant sodium citrate was added to the whole blood, the mass of which accounted for 1% of the whole blood mass. The whole blood was centrifuged at 120g for 15 minutes at room temperature to remove the red blood cells and prepare concentrated platelets.

[0112] A commonly used activation treatment method in clinical practice, a mixture of thrombin and calcium ions (finally 100 U / mL thrombin and 22.8 mM calcium ions) was added and incubated at 37°C for 30 min to prepare activated platelet-rich plasma using thrombin + calcium.

[0113] Comparative Example 4

[0114] The preparation method of this comparative example is the same as that of Example 3, except that the power of the ultrasonic treatment is 0.8 W, referred to as PRP (US) 0.8W.

[0115] Comparative Example 5

[0116] In this comparative example, the PRP product was prepared by the existing ultrasonic water bath method, referred to as PL (US1).

[0117] Comparative Example 6

[0118] In this comparative example, the existing ultrasonic probe insertion method was used to prepare the PRP product, referred to as PL (US2).

[0119] Comparative Example 7

[0120] The other steps of this comparative example are the same as those of Example 3, except that the ultrasonic power is 0.2 W, and the obtained product is referred to as PRP (US) 0.2W.

[0121] Comparative Example 8

[0122] The other steps of this comparative example are the same as those of Example 3, except that the ultrasonic power is 0.5 W, and the obtained product is referred to as PRP (US) 0.5W.

[0123] Comparative Example 9

[0124] The other steps of this comparative example are the same as those of Example 3, except that the ultrasonic power is 1.0 W, and the obtained product is referred to as PRP (US) 1.0W.

[0125] Comparative Example 10

[0126] The other steps of this comparative example are the same as those of Example 3, except that the ultrasonic power is 1.5 W, and the obtained product is referred to as PRP (US) 1.5W.

[0127] Figure 1 This is a graph of plasma after the first ultrasonic treatment in Comparative Example 1 of the present invention. Figure 2 This is a graph of plasma after the first ultrasonic treatment in Example 1 of the present invention.

[0128] from Figure 1 and Figure 2 It can be seen that when the probe gel coupling agent thickness is not appropriate, the plasma obtained contains aggregated platelet clumps, while when the gel coupling agent thickness is appropriate, there is no obvious platelet aggregation.

[0129] Figure 3 Schematic diagram comparing the PF4 content in PRP obtained in Example 1 and Example 2. As can be seen from the figure, after the first ultrasonic treatment and then culture, the content of the active substance PF4 produced and secreted by platelets is higher.

[0130] Figure 4 The following are SEM images of the PRP products obtained in Example 1, Comparative Example 2, and Comparative Example 3 of the present invention. The images show that the platelets obtained in Example 1 exhibit partial platelet activation and fragmentation. The platelets obtained in Comparative Example 2 appear flat and flake-like, with the platelets also being fragmented. The platelets obtained in Comparative Example 3 exhibit extended pseudopodia and interconnected structures, indicating activation.

[0131] Figure 5 Schematic diagram comparing the maximum platelet aggregation rates in the PRP products obtained in Example 1, Comparative Example 2, and Comparative Example 3 of the present invention. Figure 5 This is the result of a platelet aggregation test, the most classic and effective test for monitoring platelet function. Platelets lose their ability to aggregate after lysis. As can be seen in the figure, lysed platelets lack platelet aggregation.

[0132] Figure 6 Schematic diagram comparing the platelet aggregation ability of the PRP products obtained in Examples 3, 5, 8, and Comparative Examples 7 to 10 of the present invention and the untreated PRP products. Schematic diagram comparing the flow cytometry assay for the activation marker CD62P. The ultrasonic treatment power is 0.2 to 1 W / cm 2 The platelet aggregation function of the PRP product will not be affected within the range, and the platelets will survive in a biologically active state, at which time they will continue to produce and release biologically active substances. With the increase of ultrasonic treatment power, 1.5-2.5w / cm 2The platelet aggregation function in PRP treated with US decreases with increasing power, which is reflected in an increase in the lysis ratio and an increase in the release of bioactive substances stored in the platelets themselves (the bioactivity of the platelets gradually decreases, and the ability to continuously synthesize and release bioactive substances will gradually decrease).

[0133] Figure 7 The figure is a comparative diagram of the platelet activation marker CD62P flow cytometry test in the PRP products obtained in Examples 3, 5, 8, and Comparative Examples 7 to 10 of the present invention and the untreated PRP products. The platelet activation CD62P flow cytometry test is considered to be the "gold standard" test for platelet activation detection. CD62, also known as P-selectin, is a membrane glycoprotein of platelet α-granules. It is only expressed in very small amounts on the surface of the resting platelet membrane. When the platelets are activated, the α-granule membrane glycoprotein in the platelets rapidly fuses with the cell membrane through an open duct system and is the most specific marker for identifying activated platelets. Platelet activation releases active substances. This indicator indicates the activation state of platelets after treatment by the present invention, and predicts the degree to which they release specific active substances. As can be seen from the figure, the ultrasonic treatment power is between 0.2 and 1 W / cm 2 The platelet activation ratio of PRP products is low, less than 20%. 2 The platelet activation rate in the PRP product increased sharply, with the activation rate exceeding 30%.

[0134] Figure 8 This is a schematic diagram comparing the growth factor content in the PRP products obtained in Example 1 (PRP (US) 0.8+2W), Comparative Example 2 (PL), Comparative Example 3 (PRP (AA)), Comparative Example 4 (PRP (US) 0.8W), and Example 3 (PRP (US) 2W). a is the PF4 content, b is the FGF content, and c is the TGFβ content. As can be seen from the figure, compared with the PRP products obtained in the comparative examples, the PRP products obtained by the treatment method of the present invention have higher growth factors PF4 and TGFβ. β 1. The FGF content has obvious advantages.

[0135] Among them, PF4 can regulate the growth of fibroblasts and the synthesis of collagen. In the repair process after tissue damage, the proliferation of fibroblasts and the synthesis of collagen are crucial for wound healing. PF4 has the potential to reduce inflammatory responses and improve cognitive function in the elderly. PF4 also has certain antibacterial and antiviral effects, which can inhibit the growth and reproduction of certain pathogenic microorganisms and help the body resist infection. During the wound healing process, TGFβ1 can stimulate the migration of fibroblasts and epithelial cells; it can also promote the migration of vascular endothelial cells and participate in the angiogenesis process. This is crucial for the formation of new blood vessels at the wound site and the provision of oxygen and nutrients for tissue repair; it can also reshape tissue structure by regulating the synthesis and degradation of the extracellular matrix. FGF can promote the proliferation and differentiation of fibroblasts, stimulate the synthesis of collagen and elastin, and thus accelerate the wound healing process; FGF can promote the migration and differentiation of epidermal cells, enhance the barrier function of the skin, promote the production of collagen and elastic fibers, and enhance the support of the skin; FGF can regulate the function of melanocytes, inhibit the synthesis of melanin, and thus reduce the formation of spots; FGF can activate hair follicle stem cells, causing them to enter a state of proliferation and differentiation, thereby promoting hair growth.

[0136] It can be seen from the above test results that the preparation method of the present invention significantly improves the excretion of the above cytokines compared with the traditional preparation method, indicating that the PRP product obtained by the preparation method of the present invention can be used for anti-aging, promoting wound healing and other tissue repair and regeneration processes.

[0137] Figure 9 Schematic diagram comparing platelet metabolites in the PRP products obtained in Example 1, Comparative Example 2, and Comparative Example 3. As can be seen from the figure, compared with PRP products obtained by traditional activation or freeze-thaw treatment, the PRP products obtained by the preparation method of the present invention have significantly upregulated metabolites with anti-inflammatory, anti-aging, and tissue repair and regeneration properties, such as α-ketoglutarate, taurine, and spermidine.

[0138] Alpha-Ketoglutarate (AKG) is a key intermediate metabolite in the tricarboxylic acid cycle (TCA cycle), widely involved in energy metabolism, amino acid synthesis, and epigenetic regulation. In particular, as a cosubstrate for dioxygenases (such as TET enzymes and histone demethylases), it influences DNA demethylation and histone modification, thereby regulating gene expression. AKG also scavenges reactive oxygen species (ROS), maintains mitochondrial function, reduces oxidative damage, and activates the Nrf2 pathway, enhancing cellular antioxidant defenses. It is currently commonly used as an anti-aging supplement, sports supplement, mitochondrial support, or antioxidant. AKG has been used clinically to support enteral nutrition in patients undergoing surgery or trauma to promote recovery. Taurine: By maintaining cell membrane stability and regulating intracellular calcium concentration, it protects cells from osmotic stress. It also neutralizes free radicals, reduces oxidative stress, and protects cell membranes and mitochondrial function. Based on the above mechanisms, taurine, either alone or in combination, is often used to treat congestive heart failure, angina pectoris, and as an adjunctive treatment for viral myocarditis. Taurine can alleviate nerve damage caused by hyperglycemia and is used as an adjunct to prescription medications for diabetic neuropathy. Taurine also acts as a neurotransmitter or modulator, affecting GABA receptor activity and stabilizing neuronal excitability. It is currently widely added to infant formula to mimic breast milk composition and support nervous system development. Taurine plays a key role in vascular, neural, and metabolic processes. Spermidine activates autophagy (e.g., inhibits the mTOR pathway), clearing damaged cells and delaying aging. It also participates in DNA stabilization, gene expression regulation, and epigenetic regulation, thus exerting anti-aging effects and protecting the nervous and cardiovascular systems. Spermidine is currently commonly used as a dietary supplement (in the anti-aging field) and as a primary anti-aging ingredient in anti-aging serums and creams. 12-Hydroxyeicosatrienoic acid (12-HETrE): As a vasoactive molecule, 12-HETrE may induce vasodilation and regulate local blood flow by activating ion channels (such as K+ channels). It also synergizes with prostaglandins (PGI2) to antagonize the vasoconstrictive effects of thromboxane A2 (TXA2). Animal models have shown that 12-HETrE can alleviate hypertension and atherosclerosis, with mechanisms involving improved endothelial function and reduced oxidative stress. At low concentrations, 12-HETrE inhibits neutrophil chemotaxis and the release of inflammatory factors (such as IL-6 and TNF-α), demonstrating anti-inflammatory properties. At high concentrations, 12-HETrE may promote inflammatory responses through ROS generation, exhibiting a concentration-dependent biphasic effect. Therefore, 12-HETrE participates in epidermal barrier repair, and topical application may alleviate dermatitis (such as atopic dermatitis). The above test results indicate that the PRP product prepared by the present method possesses anti-inflammatory properties and can be used as an anti-aging and tissue repair and regeneration agent.

[0139] Figure 10Schematic diagram comparing the cell proliferation after co-culturing human keratinocytes Hacat using the PRP products of Examples 3, 5, 8, Comparative Examples 7-10 and untreated. As can be seen from the figure, the ultrasonic power is greater than 2.0 W / cm 2 After that, its ability to promote cell proliferation increased significantly, and the ultrasound power was 2.0 W / cm 2 ~3.0 W / cm 2 The ability to promote cell proliferation is significantly higher than that of other treatment powers.

[0140] Figure 11 This is a schematic diagram comparing the cell proliferation of human keratinocytes Hacat after co-culture of the PRP products obtained in Examples 1, 2, 3, 5, and 6 of the present invention. (a) shows the proliferation of human keratinocytes Hacat promoted by the PRP products of Examples 1, 2, 3, and 6, and untreated; (b) shows the proliferation of human keratinocytes Hacat promoted by the PRP products of Examples 1, 5, 6, and 6, and untreated. It can be seen from the figure that the preparation method of the present invention has significantly enhanced the proliferation of human keratinocytes Hacat compared to existing methods.

[0141] Figure 12 This figure compares the cell viability of Example 1 (PRP (US) 0.8+2W), Example 3 (PRP (US) 2W), and untreated PRP products after co-culture with human keratinocytes (Hacat). As can be seen, the PRP products prepared by the present invention exhibit significant cell proliferation-promoting properties, with Comparative Example 1 achieving the highest performance.

[0142] Figure 13 This is a schematic diagram comparing the viability of PRP products obtained in Example 1, Comparative Example 2, and Comparative Example 3 of the present invention after co-culture with primary mouse fibroblasts. Figure 14 Schematic diagram comparing the migration ability of PRP products obtained in Example 1, Comparative Example 2, and Comparative Example 3 after co-culture with mouse primary fibroblasts. Figure 13 and Figure 14 The results show that the PRP product prepared by the preparation method of the present invention has significant advantages in promoting the proliferation and migration of primary mouse fibroblasts compared with the control example.

[0143] Figure 15This figure compares the therapeutic effects of the PRP products obtained in Example 1, Comparative Example 2, and Comparative Example 3 on deep second-degree burn wounds in rats. A rat burn model was first prepared, and then the burn wounds were treated daily with the products obtained in Example 1, Comparative Example 2, and Comparative Example 3 (topical treatment, using methods similar to existing PRP products). Fourteen days after treatment, the PRP products obtained by the present invention were significantly more effective than those obtained by existing methods, demonstrating excellent therapeutic efficacy.

[0144] Ultrasonic therapeutic devices utilize low-power pulsed ultrasound waves for therapeutic purposes through their thermal, mechanical, and physicochemical effects. They are suitable for treating soft tissue injuries, osteoarthritis, and other conditions. Commonly used ultrasonic therapeutic devices have a power range of approximately 0.2-3 W / cm², with probe sizes of 1, 2, 4, 5, and 10 square centimeters. Compared to ultrasonic cell disrupters (100-500 W) commonly used for platelet lysis, these devices are considered incapable of sufficiently lysing platelets in PRP in large quantities. Consequently, their use in preparing platelet lysates and their properties and uses have remained largely unexplored. However, the present invention utilizes a unique low-intensity pulsed ultrasound, further combined with a specific ultrasound regime, to produce a PRP product with both activation and lysis characteristics. Compared to existing methods, the PRP product produced by this method produces and releases active substances (growth factors, metabolites, etc.) that promote tissue repair and regeneration, as well as anti-aging effects. It exhibits remarkable results in promoting cell proliferation, cell migration, and wound healing. And because the ultrasonic therapeutic device is portable and easy to operate, it can be used as an in situ treatment mode with controllable time and space for in vivo injection, and can also be used as an in vitro PRP treatment mode to obtain high-quality platelet-rich plasma PRP products that are anti-aging and promote tissue repair and regeneration.

Claims

1. A method for preparing platelet-rich plasma for anti-aging and promoting tissue repair and regeneration, characterized in that: The following steps are involved: Platelet-concentrated plasma is obtained and treated with low-intensity pulsed ultrasound. The platelet-concentrated plasma is placed in a well culture plate using an ultrasound therapy device for ultrasound treatment. The probe of the ultrasound therapy device is located at the bottom of the well plate, with the center of the probe facing the center of the well. A 0.5-1 cm gel coupling agent is placed between the probe and the well plate. The processing condition is one of the following processing methods: 1) Ultrasonic treatment power is 2~2.5 W / cm 2 , the pulse period was 50%, the ultrasonic treatment time was 3 min, and the ultrasonic frequency was 1 MHz; 2) First, perform the first ultrasonic treatment: use 0.8 W / cm 2 The pulse period was 50%, the ultrasonic treatment lasted for 3 min, and the ultrasonic frequency was 1 MHz. The plasma after the first ultrasonic treatment was placed at 37 °C in air containing 5% CO2 for 1 h. Then the second ultrasonic treatment was carried out: 2.0 W / cm 2 , pulse period was 50%, ultrasonic treatment was 3 min, and ultrasonic frequency was 3 MHz; The desired platelet-rich plasma can be obtained after ultrasonic treatment.

2. The method for preparing platelet-rich plasma for anti-aging and promoting tissue repair and regeneration according to claim 1, characterized in that: The platelet concentrated plasma preparation process is as follows: Anticoagulant is added to the blood and concentrated platelet plasma is obtained after centrifugation; the anticoagulant is sodium citrate anticoagulant, and the added volume accounts for 1% of the blood volume.

3. The method for preparing platelet-rich plasma for anti-aging and promoting tissue repair and regeneration according to claim 1, characterized in that: The platelet-rich plasma contains active substances that are beneficial to tissue repair and regeneration and anti-aging, and the active substances include metabolites and growth factors; the metabolites include α-ketoglutaric acid, taurine, and spermidine.

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