Injectable growth factor hydrogel as well as preparation method and application thereof

Abstract

The invention provides injectable growth factor hydrogel as well as a preparation method and application thereof, and relates to the technical field of medical materials. The injectable growth factor hydrogel is developed by taking glycyrrhizic acid (GA) and dopamine modified hyaluronic acid (HA-DA) as a biological matrix material and taking baicalein A (OA) and a platelet-derived growth factor (PDGF-BB) as bioactive functional components. The invention aims to solve the technical problems of non-lasting growth factor release, difficult hydrogel injection, single endometrial repair effect and the like in the existing treatment method, can promote tissue repair and fertility recovery, and has a remarkable clinical application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medical materials, and particularly relates to an injectable growth factor hydrogel and a preparation method and application thereof. BACKGROUND

[0002] Intrauterine adhesion (IUA) refers to the adhesion of the endometrium, which is usually caused by surgical trauma, infection, post-abortion sequelae or other factors. IUA seriously affects the reproductive function of women, often leading to infertility, miscarriage and premature birth and other problems. The treatment of intrauterine adhesion usually relies on surgical intervention, however, surgical treatment may not completely restore the structure and function of the endometrium, and the treatment effect is often limited.

[0003] The existing treatment methods mainly include physical treatment, hormone treatment and local drug treatment. Although these methods can alleviate symptoms to some extent, there are still shortcomings such as unstable curative effect, long treatment time and non-persistent treatment effect. As a material with good biocompatibility, hydrogel has been widely used in the fields of tissue engineering, drug delivery and regenerative medicine in recent years. Hydrogel can be used as a drug carrier for slow release, thereby continuously exerting a therapeutic effect at the target site.

[0004] Glycyrrhiza as a classic medicinal and edible traditional Chinese medicine, its core active ingredient glycyrrhizic acid (GA) has multiple physiological functions, including antibacterial, anti-inflammatory, antiviral and anticancer. These characteristics make it have application value in cell damage, cell regeneration and tumor treatment. The molecular structure of glycyrrhizic acid contains a hydrophobic triterpene glycoside base (18β-glycyrrhizinic acid) and two hydrophilic glucuronic acids. Studies have shown that glycyrrhizic acid has self-assembly properties in aqueous solution: through non-covalent interaction, glycyrrhizic acid can form nanofibers. When the concentration is more than 0.3wt%, these nanofibers further interweave to form a supramolecular hydrogel with a three-dimensional network structure.

[0005] Oroxylin A (OA) has been shown to regulate various biological processes and exert its effects by modulating the production of reactive oxygen species (ROS) and regulating signaling pathways involved in inflammation and cell survival. Studies have shown that OA has therapeutic potential in various inflammatory and fibrotic diseases (such as liver fibrosis). Notably, OA has also been shown to inhibit macrophage pyroptosis through the ROS pathway, thereby reducing uterine fibrosis. These findings highlight the potential of OA as an IUA treatment drug, particularly in relation to endometrial regeneration.

[0006] Growth factors, especially platelet-derived growth factor BB (PDGF-BB) and other factors that promote cell proliferation, angiogenesis and tissue repair, play an important role in endometrial repair. Growth factors can stimulate cell proliferation and migration, promote the formation of new blood vessels, and accelerate tissue regeneration, and are important components for repairing the endometrium. However, the direct application of traditional growth factors faces problems such as rapid degradation in the body, short half-life, and short-lasting local biological effects.

[0007] At present, although some studies have reported growth factor-containing hydrogel materials, there is still room for further optimization in terms of injectability, degradability, long-term stability, and endometrial repair. The present application uses glycyrrhizic acid (GA) and dopamine-modified hyaluronic acid (HA-DA) as a biomatrix material, and uses oridonin A (OA) and platelet-derived growth factor BB (PDGF-BB) as bioactive functional components to develop a new type of injectable growth factor hydrogel, which can effectively improve the limitations of existing treatment methods and provide a new treatment platform for IUA and other endometrial injury repair. SUMMARY

[0008] In view of the above shortcomings and deficiencies of the prior art, the present application provides an injectable growth factor hydrogel and its preparation method and application, aiming to solve the technical problems of short-lasting growth factor release, difficult injection of hydrogel, and single endometrial repair effect in existing treatment methods.

[0009] To achieve the above-mentioned purpose, the present application provides the following technical solutions: A preparation method of an injectable growth factor hydrogel, characterized in that it comprises the following steps: Step one, synthesis of dopamine-modified hyaluronic acid: Dissolve sodium hyaluronate in phosphate buffer, then add dopamine hydrochloride to obtain reaction solution one, stir, dialyze reaction solution one with ultrapure water for 2-3 days and freeze-dry to obtain HA-DA; Step two, synthesis of glycyrrhizic acid solution: Add glycyrrhizic acid powder to phosphate buffer to obtain reaction solution two, ultrasonically treat reaction solution two to obtain a glycyrrhizic acid suspension, heat in an oil bath at 70-80°C to obtain a GA solution; Step three, preparation of precursor solution: Add OA to the GA solution to obtain precursor solution one, dissolve HA-DA obtained in step one in water to obtain HA-DA solution, and then add PDGF-BB to the HA-DA solution to obtain precursor solution two; Step four, self-assembly to form injectable growth factor hydrogel: The precursor solution one and the precursor solution two with a volume ratio of 0.5-1:1-2 are mixed at 75-85℃ to form a mixed solution, and after the mixed solution is cooled to room temperature, self-assembly to form an injectable growth factor hydrogel, namely a BB / OA / GHD hydrogel.

[0010] Further, in the step one, the mass of the sodium hyaluronate is 4.5-7g, the mass of the dopamine hydrochloride is 0.5-1g, the pH of the phosphate buffer is 5.0, and the volume is 100-150mL; the specific step of stirring is that the reaction solution one is stirred at 20-30℃ for 8-12 hours.

[0011] Further, in the step one, the mass of the sodium hyaluronate is 5g, the mass of the dopamine hydrochloride is 0.5g, the pH of the phosphate buffer is 5.0, and the volume is 100mL.

[0012] Further, in the step two, the mass of the glycyrrhizic acid powder is 0.5-0.8g, the pH of the phosphate buffer is 7.4, the volume is 100mL, the reaction condition of the ultrasonic treatment is that the power is 80-150W, the working time is 2 seconds, the intermittent time is 8 seconds, and the cumulative ultrasonic treatment time is 2 minutes, and the concentration of the GA solution is 0.5-0.8wt%.

[0013] Further, in the step two, the mass of the glycyrrhizic acid powder is 0.5g.

[0014] Further, in the step three, the mass of the OA is 0.3-0.8g, the volume of the GA solution is 100mL, the mass of the PDGF-BB is 0.3-0.8g, and the volume of the HA-DA solution is 100mL.

[0015] Further, in the step three, the mass of the OA is 0.5g, and the mass of the PDGF-BB is 0.5g.

[0016] The application further provides an injectable growth factor hydrogel prepared by the preparation method.

[0017] The application further provides an application of the injectable growth factor hydrogel in preparing a medicine for treating intrauterine adhesion and endometrial injury.

[0018] The application has the following beneficial effects: The present application develops an injectable growth factor hydrogel by taking glycyrrhizic acid (GA) and dopamine-modified hyaluronic acid (HA-DA) as a biological matrix material, and taking oridonin A (OA) and platelet-derived growth factor BB (PDGF-BB) as a bioactive functional component, which can promote tissue repair and fertility recovery, and has a significant clinical application prospect.

[0019] The injectable growth factor hydrogel can effectively solve the problems of unstable growth factor release rate and single repair effect in the existing treatment method, and provides an injectable growth factor hydrogel, which has a wide application prospect, especially in the treatment of intrauterine adhesion and endometrial injury. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 It is an internal morphology scanning electron micrograph of the injectable growth factor hydrogel of the present application; Figure 2 It is a nuclear magnetic resonance spectrogram; Figure 3 It is an ultraviolet spectrogram; Figure 4 It is an infrared spectrogram; Figure 5 It is a gel forming real object diagram of the injectable growth factor hydrogel of the present application, wherein A is: a diagram of the initial sol state, and B is: a diagram of the gel state after 1 minute; Figure 6 It is an injectability characterization diagram of the injectable growth factor hydrogel of the present application, wherein A is: a schematic diagram of a growth factor extruding needle, and B is: a schematic diagram of extruding to form a specific shape "XY"; Figure 7 It is a biocompatibility characterization diagram of the injectable growth factor hydrogel of the present application, wherein A is: a schematic diagram of continuous degradation of the injectable growth factor hydrogel within about 8 days after subcutaneous injection in SD rats, B is: a curve diagram of continuous degradation of the injectable growth factor hydrogel within about 8 days after subcutaneous injection in SD rats, and C is: a schematic diagram of subcutaneous tissue changes within 12 days after subcutaneous injection of the injectable growth factor hydrogel in SD rats; Figure 8 It is a characterization diagram of the influence of the injectable growth factor hydrogel of the present application on the proliferation of endometrial stromal cells in vitro, wherein A is: a schematic diagram of cell counting kit-8 detection of cell viability, B is: a schematic diagram of live cell staining to observe cell proliferation, C is: a quantitative analysis diagram of 5B-C live cell staining to observe cell proliferation, and D is: a result diagram of active oxygen level detection; Figure 9 It is a rat uterine tissue HE staining diagram; Figure 10Figure for characterization of the influence of the injectable growth factor hydrogel of the present application on the endometrial thickness and glands of rats, wherein A is: a schematic diagram of quantitative analysis of endometrial thickness in each group; and B is a schematic diagram of quantitative analysis of the number of glands in each group; Figure 11 Figure for Masson's staining of uterine tissue of rats; Figure 12 Figure for characterization of the number of reproductive embryos and young rats of the injectable growth factor hydrogel of the present application, wherein A is: a figure for the number of embryos in each group, and B is a figure for the number of fetuses in each group; Figure 13 Figure for the sustained release amount of OA and PDGF-BB in the injectable growth factor hydrogel of the present application, wherein A is: a figure for the sustained release amount of OA, and B is a figure for the sustained release amount of PDGF-BB. DETAILED DESCRIPTION

[0021] Glossary: GHD hydrogel: a hydrogel formed by self-assembly of glycyrrhizic acid (GA) and dopamine-modified hyaluronic acid (HA-DA) as a biological matrix material, which is named as GHD hydrogel according to the first letters of the components.

[0022] BB / OA / GHD hydrogel: a hydrogel formed by self-assembly of glycyrrhizic acid (GA) and dopamine-modified hyaluronic acid (HA-DA) as a biological matrix material, and baicalein A (OA) and platelet-derived growth factor BB (PDGF-BB) as bioactive functional components, which is named as BB / OA / GHD hydrogel according to the first letters of the components.

[0023] OA / GHD hydrogel: a hydrogel formed by self-assembly of glycyrrhizic acid (GA) and dopamine-modified hyaluronic acid (HA-DA) as a biological matrix material, and baicalein A (OA) as a bioactive functional component, which is named as OA / GHD hydrogel according to the first letters of the components.

[0024] In order for those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be described clearly and completely in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor should fall within the scope of protection of the present application. It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict.

[0025] Embodiment 1: The embodiment provides an injectable growth factor hydrogel, taking GA and HA-DA as a biomatrix material and taking OA and PDGF-BB as bioactive functional components, so as to obtain the injectable growth factor hydrogel.

[0026] The preparation method of the injectable growth factor hydrogel comprises the following steps: Step 1: Synthesis of dopamine-modified hyaluronic acid 4.5g of sodium hyaluronate is dissolved in 100mL of phosphate buffer (pH=5.0), then 0.5g of dopamine hydrochloride is added to obtain a reaction solution one, the reaction solution one is stirred at 20℃ for 10 hours, then the reaction solution one is dialyzed with ultrapure water for 2 days and freeze-dried to obtain 5g of powder-like dopamine-modified hyaluronic acid, which is abbreviated as HA-DA.

[0027] Step 2: Synthesis of glycyrrhizic acid solution 0.5g of glycyrrhizic acid (GA) powder is added to 100mL of phosphate buffer (pH=7.4) to obtain a reaction solution two, then the reaction solution two is ultrasonically treated (120W, working for 2 seconds and intermittent for 8 seconds) for 2 minutes to uniformly disperse the glycyrrhizic acid powder, to obtain a glycyrrhizic acid suspension, and the glycyrrhizic acid suspension is placed in an 80℃ oil bath to heat to form a clear solution, and the clear solution is 0.5wt% glycyrrhizic acid solution, which is abbreviated as GA solution.

[0028] Step 3: Preparation of precursor solution 0.5g of ochracean A (OA) is added to 100mL of GA solution to obtain a GA solution with 0.5wt% of OA loaded, which is abbreviated as precursor solution one. 0.5g of powder-like HA-DA obtained in step one is dissolved in 100mL of water to obtain a 0.5wt% HA-DA solution, and 0.5g of platelet-derived growth factor BB (PDGF-BB) is added to 100mL of HA-DA solution to obtain a HA-DA solution containing 0.5wt% of PDGF-BB loaded, which is abbreviated as precursor solution two.

[0029] Step 4: Self-assembly to form an injectable growth factor hydrogel The precursor one solution and the precursor two solution in a volume ratio of 1:2 are mixed at 85℃ to form a mixed solution, and after the mixed solution is cooled to room temperature, the injectable growth factor hydrogel, i.e., the BB / OA / GHD hydrogel, is self-assembled.

[0030] Embodiment 2: The difference from embodiment 1 is that, except that OA and PDGF-BB are not added in step three, the other steps are the same as those in embodiment 1, so as to obtain a GHD hydrogel. Example 3: The difference between Example 3 and Example 1 is that, in addition to not adding PDGF-BB in step three, the remaining steps are the same as Example 1, and OA / GHD hydrogel is obtained; Physicochemical property characterization: 1. Scanning electron microscope analysis: The injectable growth factor hydrogel obtained in Example 1 (i.e., BB / OA / GHD hydrogel) was frozen and freeze-dried using a freeze dryer. After freeze-drying, the internal morphology of the BB / OA / GHD hydrogel obtained in Example 1 was observed using a JEOL JSM-6010LA scanning electron microscope at an acceleration voltage of 15 kV, as shown in Figure 1 .

[0031] The scanning electron microscope results show that the internal structure of the BB / OA / GHD hydrogel presents a clear loose porous structure, with a pore diameter of about 10 μm, which is conducive to the transport of nutrients and the removal of metabolic waste.

[0032] 2. Nuclear magnetic resonance spectroscopy analysis: A Bruker AVANCE III 500 nuclear magnetic resonance spectrometer was used to verify the coupling of dopamine and hyaluronic acid (HA). The dopamine-modified hyaluronic acid (HA-DA) obtained in step one of Example 1 was dissolved in heavy water (D2O), and the proton peak was analyzed, focusing on the peak at 2.86 ppm corresponding to the catechol group of dopamine, as shown in Figure 2 .

[0033] 3. Ultraviolet spectrum analysis: A Lambad 800 spectrophotometer was used to collect the ultraviolet-visible spectrum of the dopamine-modified hyaluronic acid (HA-DA) obtained in step one. Hyaluronic acid-dopamine (HA-DA) has a characteristic absorption peak at 285 nm (attributed to the aromatic ring of dopamine), proving its successful synthesis, as shown in Figure 3 .

[0034] 4. Infrared spectrum analysis A Nicolet AVATAR 360 FT-IR spectrometer was used to obtain the Fourier transform infrared spectrum of the BB / OA / GHD hydrogel obtained in Example 1 to analyze hyaluronic acid-dopamine (HA-DA). The shift of the characteristic peak in the spectrum and the appearance of a new peak indicate that the BB / OA / GHD hydrogel is crosslinked, as shown in Figure 4 .

[0035] Sol-gel transition and gelation time The sol-gel transition process was evaluated by inverted test tube method. The precursor solution one and the precursor solution two were mixed at 85°C, then inverted test tube at 37°C, and the gelation time was recorded when the BB / OA / GHD hydrogel was stable and stayed at the bottom of the test tube. The process was completed within about 1 minute under physiological conditions.

[0036] The sol-gel transition process of the BB / OA / GHD hydrogel was visually evaluated by inverted test tube method. Under physiological conditions, the BB / OA / GHD hydrogel quickly formed a stable and transparent gel within about 1 minute and stayed at the bottom of the test tube, as shown in Figure 5

[0037] Application Example 1 injectability evaluation: 1. Injectability test: The injectable growth factor hydrogel (BB / OA / GHD hydrogel) prepared in Example 1 (150 μL) was loaded into a 1 mL syringe, and whether the BB / OA / GHD hydrogel could be smoothly pushed through the syringe was observed to test the flowability and injectability of the BB / OA / GHD hydrogel in the syringe. This test was mainly used to evaluate the performance of the BB / OA / GHD hydrogel in different specifications of syringes to ensure that it did not block during injection.

[0038] 2. Gelation characteristics after injection: The BB / OA / GHD hydrogel was injected into a transparent mold, and the gelation process of the BB / OA / GHD hydrogel after injection was observed. The gelation time was recorded, and whether the BB / OA / GHD hydrogel could quickly gel and maintain a stable shape was observed. The focus was on whether the BB / OA / GHD hydrogel could quickly change from a liquid to a solid state after injection and form a stable support structure on the wound surface after injection.

[0039] 3. Injection path control: Using a 5 mL syringe, the injection path control ability of the BB / OA / GHD hydrogel was tested. Whether the BB / OA / GHD hydrogel could form a specific injection path was observed, and whether it could be smoothly delivered to the target area in complex sites (such as injection in the uterine cavity) was evaluated. The focus of this test was to verify the injection control of the BB / OA / GHD hydrogel and its adaptability under different operating conditions.

[0040] The test results showed that the BB / OA / GHD hydrogel could be smoothly injected in a 1 mL syringe without blocking. The BB / OA / GHD hydrogel had good flowability in the syringe, could be smoothly extruded from the needle of a 23-gauge syringe, and could form the letter "XY" by conventional injection operation, as shown in Figure 6 ​As shown, it indicates that the BB / OA / GHD hydrogel has good injectability.

[0041] The gelation property of the BB / OA / GHD hydrogel after injection can effectively support wound repair, avoiding the instability phenomenon after injection in traditional materials. The BB / OA / GHD hydrogel can provide an effective treatment for intrauterine adhesion treatment and endometrial repair, and has significant clinical application potential.

[0042] Application Example 2: Degradability evaluation: In vivo degradation experiment: The purpose of this example is to evaluate the degradability and biocompatibility of the BB / OA / GHD hydrogel prepared in Example 1 in vivo, to ensure the safety and effectiveness of the BB / OA / GHD hydrogel in endometrial repair, especially its degradation rate in vivo and interaction with surrounding tissues.

[0043] The BB / OA / GHD hydrogel prepared in Example 1 was injected 1ml subcutaneously into SD rats, and the degradation process of the BB / OA / GHD hydrogel was observed. Every 2 days (for a total of 8 days), the tissues at the injection site of the SD rats were removed for histological section analysis to observe the degradation degree of the BB / OA / GHD hydrogel and its interaction with surrounding tissues. The degradation process of the BB / OA / GHD hydrogel was recorded to evaluate its degradation window in vivo, ensuring that it provides effective repair while avoiding too fast or too slow degradation. The biocompatibility of the BB / OA / GHD hydrogel after subcutaneous injection in SD rats was evaluated, mainly by histological analysis to evaluate the tissue reaction around the injection site. At 4 days, 8 days and 12 days after injection, tissue samples at the injection site were removed for HE staining and immunohistochemical staining to observe the local inflammatory response and the compatibility of the BB / OA / GHD hydrogel with the tissue. The degree of local inflammatory response was recorded, and the degradation process of the BB / OA / GHD hydrogel and tissue repair was analyzed.

[0044] As shown in A and B of Figure 7 The BB / OA / GHD hydrogel showed a sustained degradation process of about 8 days, as shown in A and B of Figure 7As shown in FIG. 1C, it is indicated that the BB / OA / GHD hydrogel has good biocompatibility.

[0045] Application Example 3 promotes cell proliferation and regulates oxidative stress evaluation: 1. Cell culture and treatment: Human endometrial stromal cells (ESCs) were used, and the cells were seeded in 96-well plates and cultured in DMEM / F-12 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.

[0046] (1) GHD group: inject 100 uL of hydrogel without OA and PDGF-BB (i.e., inject the GHD hydrogel prepared in Example 2); (2) OA / GHD group: inject 100 uL of hydrogel containing OA but not PDGF-BB (i.e., inject the OA / GHD hydrogel prepared in Example 3); (3) BB / OA / GHD group: inject 100 uL of hydrogel containing OA and PDGF-BB (i.e., inject the BB / OA / GHD hydrogel prepared in Example 1); (4) Control group: inject 100 uL of DMEM / F-12 cell culture medium.

[0047] Detection was performed after 48 hours of cell culture.

[0048] 2. Cell proliferation evaluation (CCK-8 method): The CCK-8 kit was used to evaluate the proliferation of cells in each group. After 48 hours of culture, CCK-8 reagent was added and incubated for 1 hour, and the absorbance (OD value) was measured using a microplate reader. By comparing the OD values of each treatment group with the control group, the effect of hydrogel on cell proliferation was evaluated.

[0049] 3. Live cell staining: Live cell staining reagent (such as Calcein AM) was used for cell viability staining to observe the survival of cells in different treatment groups. The number of live cells in each group was counted by fluorescence microscopy, and the effect of hydrogel on cell growth was further evaluated.

[0050] 4. Cell proliferation and reactive oxygen species (ROS) level detection: The reactive oxygen species detection kit (DCFH-DA) was used to detect the intracellular ROS level. After 48 hours of culture, DCFH-DA probe dye was added and incubated for 30 minutes, and the fluorescence intensity of ROS was detected using a flow cytometer. By comparing the fluorescence intensity of each group, the effect of hydrogel on oxidative stress was evaluated.

[0051] The CCK-8 assay results showed that cell proliferation in the GHD-only group and the OA / GHD group was not significantly different from that in the control group. Figure 8 As shown in Figure A, GHD hydrogel and its combinations did not have a significant negative impact on cell proliferation. However, cell proliferation was significantly increased in the BB / OA / GHD group (P<0.05), indicating that the addition of PDGF-BB to the OA / GHD group significantly promoted cell proliferation. This result was further validated by live cell staining, as shown in Figure A. Figure 8 As shown in B and C. In the BB / OA / GHD group, cell number was significantly increased, while the GHD and OA / GHD groups showed no significant change compared to the control group. ROS analysis results showed no significant difference in ROS levels between the GHD group and the control group, as shown in Figures B and C. Figure 8 As shown in Figure D. However, both the OA / GHD group and the BB / OA / GHD group significantly reduced intracellular ROS levels (P<0.05), indicating that hydrogel treatment in the OA / GHD group and the BB / OA / GHD group can effectively alleviate intracellular oxidative stress and protect cells from damage.

[0052] This embodiment demonstrates that the BB / OA / GHD group can significantly promote the proliferation of human endometrial stromal cells and effectively reduce oxidative stress levels. In summary, the BB / OA / GHD hydrogel of this invention shows significant clinical potential in the application of endometrial repair and the treatment of intrauterine adhesions.

[0053] Application Example 4: Endometrial Repair Assessment 1. Animal model construction: Fifty adult female SD rats (weighing 180-200g) purchased from Beijing Vital River Co., Ltd. were divided into five groups of 10 each: Control group, GHD group, OA / GHD group, BB / OA / GHD group, and Etoh group (ethanol-induced injury group). Endometrial injury was induced in all five groups by local injection of 1ml of 95% ethanol (Etoh), and the degree of injury was observed and recorded for 14 days.

[0054] 2. Treatment and sampling: On days 7 and 14 following ethanol-induced endometrial injury, female SD rats of each age group were treated as follows: (1) Control group: No processing is performed; (2) GHD group: 1 mL of GHD hydrogel without OA and PDGF-BB (i.e., the GHD hydrogel prepared in Example 2) was injected. (3) OA / GHD group: 1 mL of GHD hydrogel containing OA without PDGF-BB was injected (i.e., OA / GHD hydrogel prepared in Example 3); (4) BB / OA / GHD group: 1 mL of GHD hydrogel containing OA and PDGF-BB was injected (i.e., BB / OA / GHD hydrogel prepared in Example 1); (5) Etoh group: 1 mL of saline was injected, and after 14 days, the animals were sacrificed and uterine tissues were removed for histological analysis.

[0055] 3. Histological analysis: The rat uteruses were removed, fixed, dehydrated, embedded, and subjected to HE staining and Masson's staining. The endometrial thickness, glandular structure, and matrix reconstruction were observed. The endometrial repair effects of different groups were observed through histological sections, and the endometrial thickness and glandular density were measured using a microscope for image analysis. Statistical analysis was performed using one-way ANOVA to compare the differences between different treatment groups.

[0056] 4. Fertility evaluation: On the 28th day after endometrial repair treatment, the number of embryos and fetuses was counted through animal dissection. Compared with the Control and Etoh groups, the effect of BB / OA / GHD hydrogel treatment on fertility recovery was evaluated. By observing the embryonic development, the recovery effect of BB / OA / GHD hydrogel treatment on fertility function was further verified.

[0057] The Etoh group showed severe endometrial damage, with a significantly thinned endometrium and lost glands. Compared with the Control group, the endometrial thickness of the Etoh group was significantly reduced, and the glands were almost lost, showing obvious tissue damage. As shown in FIG. 1A and FIG. 1B, the scale bar = 100 μm. Figure 9

[0058] The endometrium of the Etoh group showed severe gland loss and matrix tissue disorder. The GHD group partially restored the gland structure, but the repair effect was limited. The OA / GHD group showed significant repair effect, with increased gland number and gradually restored matrix tissue structure. However, the BB / OA / GHD group showed the most significant repair effect, with increased endometrial thickness, significantly improved gland density (P < 0.05), and well-repaired matrix and epithelial structures, as shown in FIG. 2A and FIG. 2B. The endometrial tissue structure of the BB / OA / GHD group was complete, with ordered gland and matrix tissues, showing the best treatment effect. Figure 10

[0059] As shown in FIG. 3A and FIG. 3B, Figure 11 Figure 11 ​​​The scale bar is 100 μm. The Etoh group showed significant collagen deposition, indicating that ethanol exposure led to fibrotic changes in the endometrial tissue, which may hinder endometrial regeneration and repair. Compared to the Etoh group, the GHD group showed less collagen deposition, suggesting that GHD hydrogel may play a protective role in collagen synthesis by modulating inflammatory pathways. The OA / GHD group showed more significant relief of collagen deposition, indicating that the combined use of OA and GHD more effectively modulates collagen synthesis. The BB / OA / GHD group showed the lowest collagen deposition, indicating that the addition of PDGF-BB further enhanced the efficacy of OA / GHD, providing stronger protection for endometrial repair and reducing collagen accumulation.

[0060] like Figure 12 As shown in Figures A and B, the number of embryos and fetuses in the Etoh group was significantly lower than that in the Control group (P<0.05), indicating that ethanol exposure leads to impaired fertility and reduced embryo viability. This is consistent with previous findings related to ethanol-induced endometrial dysfunction. In contrast, the number of embryos and fetuses in the GHD group was significantly higher than that in the ethanol group (P<0.05), showing that GHD hydrogel can improve fertility outcomes. The OA / GHD group and the BB / OA / GHD group further improved fertility outcomes, with a significant increase in the number of embryos and fetuses in both groups (P<0.05), demonstrating better fertility recovery compared to the Etoh group. In particular, the number of embryos and fetuses in the BB / OA / GHD group was close to that in the Control group, indicating that this combined treatment has significant efficacy in restoring endometrial function and promoting successful pregnancy.

[0061] This embodiment demonstrates that the BB / OA / GHD group significantly promotes endometrial repair in rats. The BB / OA / GHD group exhibited the strongest repair effect, not only restoring endometrial thickness but also significantly increasing the number of glands and the orderly structure of the stromal tissue. In contrast, the ethanol group showed significantly increased collagen deposition, leading to endometrial fibrosis and impaired fertility. In comparison, BB / OA / GHD hydrogel treatment significantly reduced collagen deposition, significantly increased the number of embryos and fetuses, restored endometrial function, and promoted fertility recovery. Histological analysis further supports the clinical potential of BB / OA / GHD hydrogel in endometrial repair. This hydrogel effectively promotes endometrial regeneration and has broad application prospects, especially in the treatment of intrauterine adhesions and fertility recovery.

[0062] Application Example 5: Evaluation of Sustained-Release Effect The release curves of OA and PDGF-BB in the hydrogel were evaluated by enzyme-linked immunosorbent assay (ELISA). Specifically, 1 mL of GHD hydrogel containing only PDGF-BB and no OA (the steps are the same as those in Example 1 except that OA is not added in step three) and 1 mL of GHD hydrogel containing only OA and no PDGF-BB (the steps are the same as those in Example 1 except that PDGF-BB is not added in step three) were respectively placed in a phosphate buffer (pH = 7.4) in a 37℃ incubator, and the cumulative release amounts of PDGF-BB and OA were respectively monitored, and the concentrations of OA and PDGF-BB in the release medium were determined at the set time points.

[0063] The sustained release of OA and PDGF-BB in the hydrogel was evaluated, and the results showed that OA was gradually released within 7 days, and the release amount at the 7th day accounted for about 95% of the total amount; PDGF-BB also showed a sustained release trend, as shown in Figure 13

[0064] In summary, the present application provides an injectable growth factor hydrogel and a preparation method thereof, which adopts a step-by-step strategy, has excellent injectability and biocompatibility, has important clinical application prospects in the treatment of intrauterine adhesion and the promotion of reproductive recovery, can effectively improve the regeneration of endometrium and embryonic development, and restore the reproductive ability.

[0065] The above-mentioned example numbers of the present application are only for description, and do not represent the advantages and disadvantages of the examples.

[0066] Those skilled in the art should understand that the purpose of the present application is to provide a relatively universal overall preparation process or technical principle, and to cover as many different applicable scenarios and conditions as possible (such as different raw material characteristics, production scale, product demand, etc.), therefore, some specific operations can be flexibly adjusted according to the situation during actual implementation, as long as the expected or the same or similar technical effects as in the embodiments of the present application can be achieved.

[0067] In the above embodiments of the present application, the description of each embodiment has its own focus, and the parts not described in detail in a certain embodiment can be referred to the related description of other embodiments. The steps in the method of the embodiments of the present application can be adjusted in sequence, combined and reduced according to actual needs. Any combination of technical features can be made, and in order to make the description simple, all possible combinations of technical features in the embodiments are not described, however, as long as the combination of technical features does not exist contradictory, it should be considered as the range disclosed in the present application.​

Claims

1. A method of preparing an injectable growth factor hydrogel, characterized by, It comprises the following steps: Step one, synthesis of dopamine modified hyaluronic acid: Dissolve sodium hyaluronate in phosphate buffer, then add dopamine hydrochloride to obtain reaction solution one, stir, dialyze reaction solution one with ultrapure water for 2-3 days and freeze-dry to obtain HA-DA; Step two, synthesis of glycyrrhizic acid solution: Add glycyrrhizic acid powder to phosphate buffer to obtain reaction solution two, ultrasonically treat reaction solution two to obtain glycyrrhizic acid suspension, heat in an oil bath at 70-80℃ to obtain GA solution; Step three, preparation of precursor solution: Add OA to GA solution to obtain precursor solution one, dissolve HA-DA obtained in step one in water to obtain HA-DA solution, then add PDGF-BB to HA-DA solution to obtain precursor solution two; Step four, self-assembly to form injectable growth factor hydrogel: Mix precursor solution one and precursor solution two in a volume ratio of 0.5-1:1-2 at 75-85℃ to form a mixed solution, and after the mixed solution cools to room temperature, self-assemble to form an injectable growth factor hydrogel, i.e. BB / OA / GHD hydrogel.

2. The method of claim 1, wherein the injectable growth factor hydrogel is prepared by, In the step one, the mass of the sodium hyaluronate is 4.5-7g, the mass of the dopamine hydrochloride is 0.5-1g, the pH of the phosphate buffer is 5.0, and the volume of the phosphate buffer is 100-150mL; the specific steps of the stirring are as follows: stirring the reaction solution one at 20-30℃ for 8-12 hours.

3. A method of preparing an injectable growth factor hydrogel according to claim 2, wherein, In the step one, the mass of the sodium hyaluronate is 5g, the mass of the dopamine hydrochloride is 0.5g, the pH of the phosphate buffer is 5.0, and the volume of the phosphate buffer is 100mL.

4. A method of preparing an injectable growth factor hydrogel according to claim 3, wherein, In the step two, the mass of the glycyrrhizic acid powder is 0.5-0.8g, the pH of the phosphate buffer is 7.4, the volume of the phosphate buffer is 100mL, the ultrasonic treatment conditions are as follows: 80-150W, working for 2 seconds, intermittent for 8 seconds, cumulative ultrasonic treatment for 2 minutes, and the concentration of the GA solution is 0.5-0.8wt%.

5. A method of preparing an injectable growth factor hydrogel according to claim 4, wherein, In the step two, the mass of the glycyrrhizic acid powder is 0.5g.

6. A method of preparing an injectable growth factor hydrogel according to claim 5, wherein, In the step three, the mass of the OA is 0.3-0.8g, the volume of the GA solution is 100mL, the mass of the PDGF-BB is 0.3-0.8g, and the volume of the HA-DA solution is 100mL.

7. A method of preparing an injectable growth factor hydrogel according to claim 6, wherein, In the step three, the mass of the OA is 0.5g, and the mass of the PDGF-BB is 0.5g.

8. An injectable growth factor hydrogel prepared by the preparation method of any one of claims 1-7.

9. Use of the injectable growth factor hydrogel of claim 8 in the preparation of a drug for treating intrauterine adhesion and endometrial injury.

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