Platelet-derived factor composite temperature-sensitive hydrogel as well as preparation method and application thereof

Abstract

The invention relates to platelet-derived factor composite temperature-sensitive hydrogel which carries an active ingredient platelet-derived factor, is derived from pig blood and has good healing promoting effect and biocompatibility, the active ingredient is wrapped by a temperature-sensitive gel matrix, can be slowly released and has temperature-sensitive characteristic, and when the temperature reaches 37 DEG C, the active ingredient is subjected to phase change and is converted into solid gel; the wound surface is effectively isolated by a formed physical barrier which is adhered and detained in the uterine cavity; the composite temperature-sensitive hydrogel is also added with high-molecular sodium hyaluronate, so that the composite temperature-sensitive hydrogel is safe and degradable, and meanwhile, the defect that a single temperature-sensitive component is low in mechanical strength and easy to corrode is overcome; the preparation method of the temperature-sensitive hydrogel is simple and low in cost; the composite temperature-sensitive hydrogel can be used for preparing drugs for in-situ treatment of intrauterine adhesion, in-situ treatment is achieved, and in-vitro degradation experiments prove that the composite temperature-sensitive hydrogel is not prone to corrosion and can stay in simulated body fluid for 14 days; the cost is low, the raw materials are safe and easy to obtain, industrial conversion is facilitated, and clinical application and popularization are facilitated.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to a platelet-derived factor composite thermosensitive hydrogel and a preparation method and application thereof. Background Art

[0002] Intrauterine adhesions (IUA), also known as Asherman syndrome, are complications of partial or complete adhesions of the uterine cavity caused by fibrous tissue hyperplasia of the uterine wall and damage to the endometrium during pregnancy. It is an important cause of female subfertility. IUA is related to uterine infection, congenital malformations, placenta previa, placenta accreta, invasive diagnostic and treatment procedures, and genetic factors. Among them, invasive diagnostic and treatment procedures are the main factors that induce IUA. In recent years, with the increase in clinical artificial abortions and curettage, the incidence of IUA has also increased year by year, and the degree of adhesion has become more serious. Therefore, the prevention and treatment of IUA is an urgent problem that needs to be solved in related disciplines such as obstetrics and gynecology and reproductive medicine.

[0003] At present, the main clinical treatment for intrauterine adhesions is hysteroscopic uterine adhesion separation (TCRA). Since the recurrence rate of IUA after surgery is about 40%, it seriously affects the reproductive prognosis of patients. Therefore, necessary auxiliary treatment measures should be used after TCRA to reduce the recurrence of adhesions.

[0004] At present, the clinical methods for preventing intrauterine adhesions and their recurrence include intrauterine devices, intrauterine adaptive balloons, estrogen, anti-inflammatory and repair-promoting drugs, and sodium hyaluronate gel for intrauterine cavity. Intrauterine devices and intrauterine adaptive balloons can reduce the recurrence rate of intrauterine adhesions to some extent, but intrauterine adhesions can cause deformation and abnormal position of the uterine cavity and cervix, which usually makes the insertion and placement of the device difficult or even may fail. At the same time, the placement of the device may shift or cause uterine perforation, severe bleeding, infection or pain. For the final result, it is also difficult for them to be biodegradable, non-toxic, and have a suitable residence time in the body. Drugs can be used to treat most endometrial injuries, and can be divided into oral, vaginal and intravenous administration according to the method of administration. Both oral and intravenous administration are systemic treatments, and the local retention rate of drugs in the uterus is low, and the therapeutic effect is limited. In addition, there is currently no clear dosage and course of treatment for the use of estrogen or other drugs such as aspirin, and there is no strict international standard for safety. Although vaginal administration can achieve targeted delivery, it can easily lead to excessive local drug concentration and produce side effects. Currently, sodium hyaluronate gel (such as Gongankang, etc.) is commonly used in clinical practice. It may have a beneficial effect on preventing intrauterine adhesions when used alone, but the gel alone is not enough to repair and prevent endometrial damage, and its lack of fluidity cannot completely adhere to the irregular endometrium. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a platelet-derived factor composite thermosensitive hydrogel and a preparation method and application thereof.

[0006] The technical solution of the present invention is:

[0007] The platelet-derived factor composite thermosensitive hydrogel consists of platelet-derived factor and thermosensitive gel matrix.

[0008] Furthermore, the thermosensitive gel matrix is ​​poloxamer P407 and sodium hyaluronate.

[0009] Furthermore, the concentration of each component is: platelet-derived factor 10-15% v / v, poloxamer P407 20-30% w / v, sodium hyaluronate 1-5% w / v.

[0010] Furthermore, the molecular weight of the sodium hyaluronate is 1000 kDa-1800 kDa.

[0011] The method for preparing a platelet-derived factor composite thermosensitive hydrogel comprises adding poloxamer P407 and sodium hyaluronate to a solution containing platelet-derived factors, and mechanically stirring the solution at a speed of 100-300 r / min and a temperature of 4° C. for 10-12 hours to obtain the platelet-derived factor composite thermosensitive hydrogel.

[0012] Furthermore, the preparation method of the solution containing platelet-derived factors is: collecting the white precipitate and white liquid after standing and centrifuging the anticoagulated whole blood of pigs, centrifuging the white precipitate and liquid again and collecting the precipitate, diluting the precipitate, freezing and thawing, ultrasonically crushing, and centrifuging it, and the supernatant is the solution containing platelet-derived factors.

[0013] The platelet-derived factor composite thermosensitive hydrogel prepared according to the above preparation method is used in the preparation of drugs for in situ treatment of intrauterine adhesions or thin uterus.

[0014] Furthermore, the drug is administered by vaginal injection.

[0015] Furthermore, the effective dosage of the drug is 3 to 5 ml.

[0016] Compared with the prior art, the present invention has at least the following advantages:

[0017] 1. The present invention relates to a platelet-derived factor composite thermosensitive hydrogel, which carries the active ingredient platelet-derived factor (PDFs) and has a good healing effect. Platelet-derived factors are a variety of growth factors produced after platelets are activated and lysed, mainly including platelet-derived growth factor, transforming growth factor, insulin-like growth factor, epidermal growth factor receptor, vascular endothelial growth factor, etc. The principle of its effective effect is to rely on the synergistic effect of the rich growth factors and cytokines it contains, which is more in line with the repair and regeneration needs than a single factor.

[0018] 2. The present invention relates to a platelet-derived factor composite thermosensitive hydrogel, the precursor of which has a certain fluidity and can adapt to complex geometric shapes to match uterine cavities of different shapes. Since endometrial wounds are often distributed deep in the folds and the individual uterine cavity morphology varies significantly, the ideal material needs to have both fluidity filling and morphology maintenance capabilities. Compared with the currently commonly used clinical anti-adhesion product Gong Ankang (cross-linked sodium hyaluronate gel), the self-developed thermosensitive hydrogel PDFs@Gel exhibits unique advantages: with precise gelation time, it flows and fills the entire mold (simulating the uterine cavity) before it changes phase to a semi-solid colloid at 37°C, perfectly matching the geometry of the mold. When the body temperature reaches 37°C, a phase change occurs to solid gel, which can adhere to and remain in the uterine cavity, forming a physical barrier that effectively isolates the wound.

[0019] 3. The present invention relates to a platelet-derived factor composite thermosensitive hydrogel, which introduces high-molecular sodium hyaluronate. It is safe and degradable, while improving the shortcomings of a single thermosensitive component, which is weak in mechanical strength and easy to dissolve, and prolongs the time of degradation and release. In vitro and in vivo experiments have shown that the composite thermosensitive hydrogel is not easily dissolved, can stay in simulated body fluids for 14 days, and is released in the rat uterus for 14 days, accurately matching the critical period of endometrial epithelial regeneration (5-10 days). The intelligent degradation characteristics avoid foreign body reactions caused by long-term retention, and the degradation products are naturally discharged through the reproductive tract, which not only ensures the embryo implantation environment in the next cycle, but also reduces the risk of secondary surgery.

[0020] 4. The platelet-derived factor composite thermosensitive hydrogel prepared by the method of the present invention can be used to prepare drugs for in situ treatment of intrauterine adhesions. The drugs can achieve in situ treatment, and the rat model verifies that the treatment effect is significant. And it belongs to a physical blending system. This non-covalent bonding composite characteristic has dual advantages: on the one hand, it avoids the risk of chemical modification of active ingredients, which is conducive to maintaining the original structure-activity relationship of the drug; on the other hand, due to its simple mixing mechanism, it significantly reduces the complexity of the production process, provides feasibility for large-scale production, and is conducive to clinical application and promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the specific embodiments of the present invention, the drawings required for use in the specific embodiments or the description of the prior art will be briefly introduced below.

[0022] Figure 1 This is a state diagram of the platelet-derived factor composite thermosensitive hydrogel for intrauterine use prepared in Example 2 of the present invention at room temperature and 37°C;

[0023] Figure 2 This is a result diagram of the gel shape adaptability of Test Example 1 of the present invention;

[0024] Figure 3 This is a graph showing the in vitro degradation time of the platelet-derived factor composite thermosensitive hydrogel in Test Example 2 of the present invention;

[0025] Figure 4 This is a graph showing the degradation, release and distribution of platelet-derived factor composite thermosensitive hydrogel in rats in test example 3 of the present invention;

[0026] Figure 5 HE staining images of uterine tissues of rats in each group after treatment in Test Example 4 of the present invention;

[0027] Figure 6 This is the safety verification of the platelet-derived factor composite thermosensitive hydrogel in Test Example 5 of the present invention. DETAILED DESCRIPTION

[0028] The present invention is further described in detail below. It is necessary to point out that the following specific implementation methods are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Technicians in this field can make some non-essential improvements and adjustments to the present invention based on the above application content.

[0029] The present invention provides a general and / or specific description of the materials and test methods used in the experiment. The test methods or test methods involved are all conventional methods unless otherwise specified; the reagents or instruments used are all conventional products available on the market, prepared or used in a conventional manner, unless the manufacturer is specified.

[0030] Sources:

[0031] Poloxamer P407, purchased from BASF, Germany;

[0032] Sodium hyaluronate: 1000 kDa-1800 kDa, purchased from Aladdin;

[0033] Rats: Female, purchased from Vital River.

[0034] Example 1 Preparation of platelet lysate (enriched with platelet-derived growth factor)

[0035] After anticoagulation, pour 2L of pig blood into a separatory funnel and let it stand for stratification (2 hours). Take the supernatant and centrifuge at 3750rpm for 10 minutes (9 up and 6 down). The precipitate is resuspended with saline and centrifuged again. After centrifugation, discard the supernatant, aspirate the white precipitate with a pipette into a new centrifuge tube, add saline to resuspend, centrifuge at 800g for 20 minutes (6 up and 1 down), take the white part (including precipitate and liquid), add appropriate amount of saline, centrifuge at 3000g for 10-20 minutes, and discard the supernatant. The precipitate is the collected platelets, which is dissolved and diluted with saline to a platelet concentration of about 3000×10 6 The enriched platelets are then placed in a -80°C refrigerator and frozen and thawed three times, then lysed using a cell disruptor at 80% power, on for 2s, off for 3s, and ultrasonic for 5min (adjusted according to the volume). The lysate is centrifuged at 8000g for 30min at 4°C to remove platelet membranes and other cell fragments, and the supernatant is a lysate rich in platelet-derived factors.

[0036] Example 2 Preparation method of platelet-derived factor composite thermosensitive hydrogel for intrauterine use

[0037] 10 mL of the platelet-derived factor-containing lysate prepared in Example 1 was filtered through a 0.22 μm filter membrane and mixed with 90 mL of double distilled water. 22 g of poloxamer P407 and 1 g of sodium hyaluronate were added thereto. The mixture was mechanically stirred at 200 r / min at 4° C. for 10 h to prepare a platelet-derived factor composite temperature-sensitive hydrogel.

[0038] The platelet-derived factor composite thermosensitive hydrogel for intrauterine use prepared in this embodiment is as follows Figure 1 As shown, it is liquid at room temperature and semi-solid gel at 37°C.

[0039] Example 3 Preparation method of platelet-derived factor composite thermosensitive hydrogel for intrauterine use

[0040] 12 mL of the platelet-derived factor-containing lysate prepared in Example 1 was filtered through a 0.22 μm filter membrane and mixed with 88 mL of double distilled water. 20 g of poloxamer P407 and 3 g of sodium hyaluronate were added thereto. The mixture was mechanically stirred at 300 r / min at 4° C. for 12 h to prepare a platelet-derived factor composite temperature-sensitive hydrogel.

[0041] Example 4 Preparation method of platelet-derived factor composite thermosensitive hydrogel for intrauterine use

[0042] 15 mL of the platelet-derived factor-containing lysate prepared in Example 1 was filtered through a 0.22 μm filter membrane and mixed with 85 mL of double distilled water. 30 g of poloxamer P407 and 5 g of sodium hyaluronate were added thereto. The mixture was mechanically stirred at 300 r / min at 4° C. for 12 h to prepare a platelet-derived factor composite temperature-sensitive hydrogel.

[0043] Test Example 1: Shape Adaptability Test

[0044] This test example takes the platelet-derived factor composite thermosensitive hydrogel prepared in Example 2 as an example to examine whether the hydrogel can adapt to complex geometric shapes to match the uterine cavity of different shapes. It is compared with Gong Ankang (cross-linked sodium hyaluronate gel), a commonly used anti-adhesion product in gynecological clinics. 3 ml of each gel is injected into a snowflake-shaped mold (4*0.8 cm, volume of about 5 ml, simulating the folds of the uterine cavity endometrium) with a syringe, and placed in a 37°C incubator. After 15 minutes, it is taken out for observation and recorded in the form of pictures. The results are as follows Figure 2 As shown in the figure, it can be seen that the temperature sensitivity and shape adaptability of the self-developed hydrogel (PDFs@Gel) can flow and penetrate into the gaps between the folds within the gelation time after injection. It is easier to achieve complete fitting of complex shapes than Gong Ankang pure colloid, and is a more ideal therapeutic material.

[0045] Test Example 2 In vitro dissolution (degradation) of platelet-derived factor composite thermosensitive hydrogel

[0046] This test example uses the thermosensitive hydrogel prepared in Example 2 as an example to conduct an in vitro dissolution experiment, with specific steps: 10 ml of hydrogel is taken into a container (the container is weighed in advance), and weighed after gelation at 37°C, and 1 ml of physiological saline preheated to 37°C is added, and the container is placed in a 37°C constant temperature shaker at 60 rpm for 12 hours and then stopped for 12 hours, and then taken out at a fixed point every 24 hours, that is, every day, for observation, and the liquid is absorbed with absorbent paper and weighed, and 1 ml of physiological saline preheated to 37°C is added. The dissolution rate is calculated based on the change in the remaining weight.

[0047] In vitro dissolution curve of platelet-derived factor-composite thermosensitive hydrogel Figure 3 As shown, the composite hydrogel takes two weeks to be completely dissolved in a simulated physiological environment.

[0048] Test Example 3: In vivo degradation, release and distribution of platelet-derived factor composite thermosensitive hydrogel

[0049] The molar ratio of Cy7 to platelet-derived factor (i.e., protein) was 10:1 for fluorescence labeling, and platelet lysate (PDFs) containing fluorescent derived factors was prepared, and then drug-loaded composite hydrogel (PDFs@Gel) was prepared. Female SD rats were anesthetized by intraperitoneal injection of 2% sodium pentobarbital, and the skin was prepared, disinfected, and the abdominal cavity of the rat was opened along the midline of the abdomen to expose the uterus. 15μL of fluorescently labeled PDFS and 150μL of fluorescently labeled PDFs@Gel were injected in situ into the right uterine cavity of the rats, respectively. Then the rats were killed after 1 day, 7 days, 14 days, and 21 days, and the rat uterus was collected. The fluorescence distribution map in the rat uterus was observed using a live imaging system.

[0050] The results are as follows Figure 4 As shown in the figure (fluorescence intensity from strong to weak is red→yellow→green→blue), the degradation and release of drugs in vivo were investigated by in vivo imaging, revealing the dynamic distribution characteristics of the two drug delivery systems in the uterine cavity. One day after administration, the PDFs group showed significant fluorescence aggregation in the right uterine cavity, while the PDFs@Gel group formed a semi-solid matrix through temperature-responsive sol-gel phase transition, showing a more lasting drug retention characteristic. By the 7th day, both groups of drugs had completed contralateral diffusion, but the PDFs@Gel group still maintained an effective drug concentration, and the three-dimensional network structure formed by its phase transition significantly slowed down the drug metabolism rate. Analysis of key time nodes showed that the fluorescence intensity of the PDFs@Gel group was stronger than that of the PDFs group on the 14th day. At this time, only a weak signal remained in the PDFs group, indicating that the traditional dosage form had been basically metabolized. It is worth noting that residual fluorescence can still be detected in the PDFs@Gel group at 21 days, which is highly consistent with its in vitro degradation and release test results. This drug delivery system exhibits dual advantages: ① The semi-solid gel formed by phase change can maintain effective coverage for 7-14 days, accurately matching the critical period of endometrial epithelial regeneration (5-10 days); ② The intelligent degradation characteristics avoid foreign body reactions caused by long-term retention, and the degradation products are naturally discharged through the reproductive tract, which not only ensures the environment for embryo implantation in the next cycle, but also reduces the risk of secondary surgery. This thermosensitive hydrogel system breaks through the limitations of traditional drug delivery modes, and its controlled release characteristics form a temporal and spatial match with the biological process of uterine injury repair, which has important therapeutic significance for promoting endometrial injury repair.

[0051] Test case 4: The therapeutic effect of platelet-derived factor-composite thermosensitive hydrogel

[0052] The tested rats were divided into 5 groups: ① NC group (normal healthy control group), ② IUA group (endometrial injury and intrauterine adhesion group), ③ PDFs group (endometrial injury and platelet-derived factor treatment group), ④ Gel group (endometrial injury and matrix gel treatment group), and ⑤ PDFs@Gel group (endometrial injury and platelet-derived factor composite thermosensitive hydrogel treatment group).

[0053] Specific methods:

[0054] (1) NC group: normal healthy rats, without treatment, only used as control;

[0055] IUA group: Sexually mature female SD rats weighing 220-280g were selected. The rats were anesthetized with 2% sodium pentobarbital, and the abdominal skin was prepared. Iodine and alcohol were added. A 1cm longitudinal incision was made on the midline of the skin 2-3cm above the urethral opening to expose the abdominal cavity. The Y-shaped uterus was removed with forceps. The endometrium was repeatedly scraped with a homemade spatula to simulate clinical curettage until the uterus became significantly thinner and lost its elasticity. The uterus was sutured with an eight-shaped suture and then inserted back into the abdominal cavity. The peritoneum was sutured continuously and the skin was sutured intermittently. After the operation, 20IU of penicillin was injected into the thigh muscle and a heating pad was used to maintain body temperature to improve the success rate of modeling.

[0056] (2) PDFs group: The endometrial injury animal model was established by mechanical curettage. Sexually mature female SD rats weighing 220-280 g were selected. The rats were anesthetized with 2% sodium pentobarbital. The abdominal skin was prepared and disinfected with iodine. A 1 cm longitudinal incision was made on the midline of the skin 2-3 cm above the urethral opening to expose the abdominal cavity. The Y-shaped uterus was removed with forceps. The endometrium was repeatedly scraped with a homemade scraper to simulate clinical curettage until the uterus became significantly thinner and lost its elasticity. The uterus was sutured in an "eight" shape. 15 μl of PDFs was injected into the uterine cavity on both sides. The uterus was stuffed back into the abdominal cavity. The peritoneum was sutured continuously and the skin was sutured intermittently. After the operation, 20 IU of penicillin was injected into the thigh muscle and a heating pad was used to maintain body temperature to improve the success rate of modeling.

[0057] (3) Gel group: The endometrial injury animal model was established by mechanical curettage. Sexually mature female SD rats weighing 220-280 g were selected. The rats were anesthetized with 2% sodium pentobarbital. The abdominal skin was prepared and disinfected with iodine. A 1 cm longitudinal incision was made on the midline of the skin 2-3 cm above the urethral opening to expose the abdominal cavity. The Y-shaped uterus was removed with forceps. The endometrium was repeatedly scraped with a homemade scraper to simulate clinical curettage until the uterus became significantly thinner and lost its elasticity. The uterus was sutured in an "eight" shape. 150 μl of Gel was injected into the uterine cavity on both sides. The uterus was stuffed back into the abdominal cavity. The peritoneum was sutured continuously and the skin was sutured intermittently. After the operation, 20 IU of penicillin was injected into the thigh muscle and a heating pad was used to maintain body temperature to improve the success rate of modeling.

[0058] (4) PDFs@PDFs group: The endometrial injury animal model was established by mechanical curettage. Sexually mature female SD rats weighing 220-280 g were selected. The rats were anesthetized with 2% sodium pentobarbital. The abdominal skin was prepared and disinfected with iodine. A 1 cm longitudinal incision was made on the midline of the skin 2-3 cm above the urethral opening to expose the abdominal cavity. The Y-shaped uterus was removed with forceps. The endometrium was repeatedly scraped with a homemade spatula to simulate clinical curettage until the uterus became significantly thinner and lost its elasticity. The uterus was sutured in an "eight-shaped" manner. 150 μl of PDFs@Gel was injected into the uterine cavity on both sides. The uterus was inserted back into the abdominal cavity. The peritoneum was sutured continuously and the skin was sutured intermittently. After the operation, 20 IU of penicillin was injected into the thigh muscle and a heating pad was used to maintain body temperature to improve the success rate of modeling.

[0059] After 14 days, the rats were euthanized and the uterine tissue was taken for H&E section staining. HE section staining: The uterine tissue was rinsed with saline three times and placed in 4% paraformaldehyde (4% The tissue samples were fixed with PFA overnight at room temperature and dehydrated with alcohol gradient, including 75% ethanol (1h) → 85% ethanol (1h) → 95% ethanol (1h) → 95% ethanol (50min) → 100% ethanol (50min) → 100% ethanol (50min); a transparent agent such as xylene was used to remove the alcohol in the tissue sample to make the tissue sample transparent, including xylene (Ⅰ) (30min) → xylene (Ⅱ) (30min); after transparency, the tissue was immersed in paraffin at 60℃-65℃ for 1h, the wax-immersed tissue sample was placed in a mold, the melted paraffin was poured in, and the paraffin was allowed to cool and solidify; after ensuring that the embedded tissue block had been fully cooled and solidified, it was fixed on the sample clamp of the microtome for slicing with a thickness of 5μm; the cut tissue slices were removed from the knife with a brush and unfolded in warm water (40℃); the slices were baked at 60℃ for 2h. Soak the sections in xylene I for 20 minutes, xylene II for 20 minutes, anhydrous ethanol I for 10 minutes, anhydrous ethanol II for 10 minutes, 95% alcohol for 5 minutes, 90% alcohol for 5 minutes, 80% alcohol for 5 minutes, 70% alcohol for 5 minutes, and finally wash with distilled water. Stain with hematoxylin staining solution for 2-10 minutes (adjust the time according to the staining results), rinse in tap water for about 10 minutes, and wash again with ultrapure water for about 5 seconds. Differentiate with hydrochloric acid ethanol differentiation solution (add concentrated hydrochloric acid to 95% ethanol to a final volume of 1%) for about 1-10 seconds (adjust the time according to the staining results), rinse in tap water for about 10 minutes. Stain with eosin staining solution for 8 seconds to 2 minutes (adjust the time according to the staining results). The sections were sequentially immersed in 95% ethanol I for 5 minutes, 95% ethanol II for 5 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, xylene I for 5 minutes, and xylene II for 5 minutes. After the slides were air-dried, neutral gum was added to seal the slides, and the slides were dried in an oven at 37°C for scanning with a slide scanner.

[0060] The staining results are as follows Figure 5 As shown: Compared with the NC group, the endometrial gland density in the IUA injury group was significantly reduced, and the endometrial thickness was lower than the normal value, showing typical pathological characteristics of intrauterine adhesions. On the 14th day after surgery, all treatment groups showed significant repair effects, among which the PDFs@Gel treatment group had the most prominent repair effect, with the number of glands restored and the endometrial thickness no longer significantly different from the normal group.

[0061] Test Example 5: Safety Verification of Platelet-derived Factor-compounded Thermosensitive Hydrogel

[0062] The red blood cells used in this test were taken from the remaining blood of women of childbearing age who had no abnormalities during routine blood tests at a gynecological clinic.

[0063] Washing red blood cells: centrifuge the anticoagulated blood at 3000 rpm for 10 minutes, discard the supernatant, wash the red blood cells with normal saline, centrifuge again at 3000 rpm for 10 minutes, discard the supernatant, repeat 3 times until the supernatant is clear.

[0064] Experimental groups: ① Positive (+) control group: take 20 μL of red blood cell sediment and add 1 ml of ultrapure water; ② Negative (-) control group: take 20 μL of red blood cell sediment and add 1 ml of normal saline; ③ PDFs group: take 20 μL of red blood cell sediment and add 1 ml of normal saline containing 6 μg PDFs; ④ Gel group: take 20 μL of red blood cell sediment and add Gel gel extract (according to the national standard of medical devices GB / T 16886, gel and normal saline were co-cultured to extract the extract); ⑤ PDFs@Gel group: take 20 μL of red blood cell sediment and add PDFs@Gel gel extract (according to the national standard of medical devices GB / T 16886, gel and normal saline were co-cultured to extract the extract).

[0065] Sample processing: The mixed solution was incubated at 37°C for 2 hours. After incubation, it was centrifuged at 3000 r / min for 10 minutes. The hemolysis was recorded in the form of pictures. The supernatant was placed in a 96-well plate and the OD value was measured at a wavelength of 540 nm using an ELISA reader.

[0066] Hemolysis rate = [(OD 样品 -OD 阴性 ) / (OD 阳性 -OD 阴性 )]×100%

[0067] It has been verified that multiple groups of biomaterials did not induce significant hemolysis in in vitro evaluations of contact with human red blood cells, and the hemolysis rate calculation results were all stable below the safety threshold of 5% (referring to the dual regulatory requirements of China's national standards GB / T 14233.2-2005 and ISO 10993-12:2002 on blood compatibility), fully meeting the stringent requirements for blood compatibility of implantable medical devices. This series of materials may effectively maintain the integrity of the red blood cell membrane structure through synergistic mechanisms such as surface hydrophilicity regulation and platelet adhesion inhibition.

[0068] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. These modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and specification of the present invention.

Claims

1. Platelet-derived factor composite thermosensitive hydrogel, characterized in that: The platelet-derived factor composite thermosensitive hydrogel consists of platelet-derived factors and a thermosensitive gel matrix.

2. The platelet-derived factor composite thermosensitive hydrogel according to claim 1, characterized in that: The thermosensitive gel matrix is ​​poloxamer P407 and sodium hyaluronate.

3. The platelet-derived factor composite thermosensitive hydrogel according to claim 2, characterized in that: The concentrations of the components are: platelet-derived factor 10-15% v / v, poloxamer P407 20-30% w / v, sodium hyaluronate 1-5% w / v.

4. The platelet-derived factor composite thermosensitive hydrogel according to claim 2 or 3, characterized in that: The molecular weight of the sodium hyaluronate is 1000kDa-1800kDa.

5. The method for preparing the platelet-derived factor composite thermosensitive hydrogel according to claim 4, characterized in that: Poloxamer P407 and sodium hyaluronate were added into a solution containing platelet-derived factors, and mechanically stirred for 10-12 hours at a rotation speed of 100-300 r / min and a temperature of 4° C. to prepare a platelet-derived factor composite thermosensitive hydrogel.

6. The preparation method according to claim 5, characterized in that: The preparation method of the platelet-derived factor-containing solution is as follows: collecting white precipitate and white liquid after anticoagulation treatment of pig blood, letting it stand and centrifuging it, centrifuging the white precipitate and liquid again and collecting the precipitate, and the supernatant after diluting, freezing and thawing, ultrasonically crushing and centrifuging the precipitate is the platelet-derived factor-containing solution.

7. Use of the platelet-derived factor composite thermosensitive hydrogel prepared by the preparation method according to claim 5 or 6 in the preparation of drugs for in situ treatment of intrauterine adhesions or thin uterus.

8. The use according to claim 7, characterized in that: The drug is administered by vaginal injection.

9. The use according to claim 8, characterized in that: The effective dosage of the drug is 3 to 5 ml.

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