A hydrogel for endometrial repair and a method of preparing the same

Abstract

The application discloses a hydrogel for endometrial repair, which is prepared from platelet-rich plasma, benzene boronic acid modified hyaluronic acid methacrylate (HAMA-PBA), phenyl-2,4,6-trimethyl benzoyl phosphonic acid lithium (LAP), calcium chloride (CaCl2) and polyvinyl alcohol (PVA). The application further provides a preparation method of the hydrogel, which comprises the following steps: mixing HAMA-PBA aqueous solution, LAP aqueous solution and CaCl2 aqueous solution to obtain a precursor solution A, dissolving PVA in PRP to obtain a PVA-PRP mixed solution as a precursor solution B, mixing the precursor solution A and the precursor solution B at a volume ratio of 1:1 to obtain PRP@HAMA-PBA-PVA hydrogel, and irradiating the PRP@HAMA-PBA-PVA hydrogel under ultraviolet light to obtain the hydrogel for endometrial repair. The hydrogel can significantly promote endometrial cell proliferation, has strong pro-angiogenic characteristics, inhibits endometrial fibrosis, and thus comprehensively repairs the endometrial microenvironment, promotes endometrial regeneration and improves embryo implantation rate and live birth rate.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to a hydrogel, specifically a hydrogel for endometrial repair and its preparation method. Background Technology

[0002] Intrauterine adhesions (IUA) are a gynecological condition characterized by varying degrees of adhesion or obstruction of the uterine cavity following endometrial damage. It is a leading cause of uterine infertility, severely affecting the receptivity of the endometrium to the embryo. IUA has a high incidence and low cure rate; the incidence after abortion surgery reaches 19%, and after hysteroscopic resection, the incidence is even higher, ranging from 20% to 62.5%. Currently, the standard surgical treatment for IUA is hysteroscopic adhesiolysis (TCRA), combined with various adjuvant therapies, such as physical barriers (intrauterine devices, intrauterine balloon devices, etc.) to prevent postoperative adhesions, and estrogen therapy to promote endometrial regeneration. Studies have shown that physical barriers alone are not effective in preventing IUA recurrence. Systemic estrogen use may have side effects such as increased risk of thrombosis and increased risk of systemic breast cancer, and its limited half-life and low effective concentration in the damaged endometrium further complicate the issue. Therefore, the prevention and treatment of intrauterine adhesions has become a major challenge in the field of reproductive medicine.

[0003] Platelet-rich plasma (PRP), due to its superior tissue regeneration capabilities, has attracted increasing attention from clinicians regarding its role in improving endometrial function. However, some clinical studies on PRP application in the endometrium have yielded inconsistent results, possibly due to the following limitations: 1) PRP itself has poor mechanical properties, making it easily metabolized and prone to breakdown. 2) PRP activation triggers a cascade release of growth factors, but the duration is short, far from sufficient for endometrial regeneration. 3) PRP cannot be injected after gelation, leading to incomplete filling of irregular lesions and inconvenience in use. Improving PRP stability, maintaining the continuous release of growth factors, and increasing intrauterine retention rates are key to enhancing PRP efficacy. In recent years, with the development of bioengineering and regenerative medicine, the combination of biomaterials and PRP therapy has been widely applied in many regenerative fields, including orthopedic regenerative therapy, skin wound repair, and hair regeneration. These biomaterials typically carry pre-activated or in vitro-unactivated PRP, but the problem of PRP cytokine burst release remains unresolved. Therefore, fully utilizing biomaterials to enable in vivo localized activation of PRP will greatly improve the limitations of PRP application, and localized activation and treatment with PRP will facilitate the widespread application of PRP in IUA treatment. Summary of the Invention

[0004] To address the aforementioned technical problems in the prior art, the present invention provides a hydrogel for endometrial repair and a method for preparing the same, which aims to solve the technical problem of poor efficacy of drug treatment for intrauterine adhesions in the prior art.

[0005] This invention provides a hydrogel for endometrial repair, which is a dual-network hydrogel for targeted activation of PRP, prepared from platelet-rich plasma (PRP), phenylboronic acid-modified hyaluronic acid methacrylate (HAMA-PBA), lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP), calcium chloride (CaCl2), and polyvinyl alcohol (PVA).

[0006] This invention also provides a method for preparing the above-mentioned hydrogel for endometrial repair, comprising the following steps:

[0007] 1) Dissolve phenylboronic acid-modified hyaluronic acid methacrylate, lithium phenyl-2,4,6-trimethylbenzoylphosphonate, and calcium chloride in PBS solution (pH 8.5). Add 5-6 g of phenylboronic acid-modified hyaluronic acid methacrylate, 0.1-0.2 g of lithium phenyl-2,4,6-trimethylbenzoylphosphonate, and 1-2 g of calcium chloride to each 100 mL of PBS solution. The resulting mixed solution is used as precursor solution A.

[0008] 2) Dissolve polyvinyl alcohol in platelet-rich plasma, add 1-2 g of polyvinyl alcohol to every 100 mL of platelet-rich plasma, and use the resulting polyvinyl alcohol-platelet-rich plasma mixed solution as precursor solution B.

[0009] 3) Precursor solution A and precursor solution B were mixed in a 1:1 volume ratio to obtain PRP@HAMA-PBA-PVA hydrogel;

[0010] 4) Place the PRP@HAMA-PBA-PVA hydrogel from step 3) under ultraviolet light for 20-30 seconds to obtain a hydrogel for endometrial repair.

[0011] Based on the characteristics of IUA and the properties of PRP, we designed and prepared an advanced injectable, porous, biodegradable dual-network targeted activation PRP-loaded hydrogel with multiple functions, including rapid gelation, self-repair, targeted activation of PRP, sustained release of growth factors, proliferation promotion, angiogenesis promotion, and anti-fibrosis. We first copolymerized hyaluronic acid methacrylate (HAMA) with a phenylboronic acid (PBA) complex to synthesize a methacrylated HAMA-PBA monomer. The polyphenolic groups provided by PBA chelate Ca... 2+PRP is used for targeted activation. Simultaneously, PRP is loaded into polyvinyl alcohol (PVA) via a physical combination mode. Subsequently, a 3D gel network is created by mixing HAMA-PBA and PVA together, utilizing the dynamic borate ester linkage between boric acid and 1,3-diol. Then, UV light can induce photocrosslinking of the HAMA polymer, forming a secondary network, thereby establishing a targeted activated PRP@HAMA-PBA-PVA hydrogel system (denoted as PRP@GEL).

[0012] This system employs a primary network constructed via dynamic borate bonds to promote Ca2+ within the gel network. 2+ The interaction between PRP and PRP promotes sustained localized activation of PRP and subsequent sustained release of growth factors, while also enhancing self-healing capabilities. A secondary network established through photocrosslinking enhances the adhesion and mechanical properties of the composite material in vivo. Ultimately, this system enables the localized activation of PRP within the damaged endometrium of intrauterine adhesions (IUA), fully leveraging the benefits of PRP to provide sustained therapeutic factors for endometrial regeneration. PRP coordinates the endometrial microenvironment through sustained release of growth factors, promoting endometrial regeneration and fertility restoration. PRP@GEL improves the endometrial microenvironment by dynamically and coordinately localizing and activating PRP within the uterine cavity, releasing growth factors from PRP, thereby contributing to endometrial regeneration and enhanced fertility. This injectable dual-network hydrogel, capable of localized activation of PRP, offers a promising new therapeutic strategy for endometrial repair in intrauterine adhesions.

[0013] We developed and produced locally activated PRP via injectable dual-network hydrogels (PRP@GEL) through dynamic borate ester bonds and photocrosslinking, facilitating the conjugation reaction of HAMA-PBA and PVA. This unique construction and structure enables locally activated PRP and sustained release of growth factors within the uterus, while simultaneously preventing the cascade release and rapid clearance of active growth factors. PRP@GEL has been shown to possess properties that promote proliferation, angiogenesis, anti-fibrosis, and comprehensive repair of the endometrial microenvironment. Cellular experiments revealed that PRP@GEL promotes endometrial cell proliferation and angiogenesis, and inhibits endometrial fibrosis by suppressing the expression of collagen deposition genes in HESCs via the TGFβ1-SMAD2 / 3 pathway. In vivo experiments confirmed that PRP@GEL hydrogel can promote endometrial regeneration and improve live birth rates. Therefore, locally activated PRP prepared via injectable dual-network hydrogels provides a novel and promising approach for the treatment of IUA and demonstrates tissue regeneration potential in various applications. In vitro experiments showed that PRP@GEL significantly promoted endometrial cell proliferation and exhibited strong pro-angiogenic properties, thereby comprehensively repairing the endometrial microenvironment. PRP@GEL inhibited endometrial fibrosis by regulating the expression of collagen deposition genes through the TGFβ1-SMAD2 / 3 pathway. Furthermore, in vivo experiments in IUA model rats treated with PRP@GEL showed that PRP@GEL significantly promoted endometrial regeneration and functional remodeling. IUA model rats treated with PRP@GEL almost completely recovered, with significantly increased embryo implantation number, litter size, and live birth rate, similar to normal rats. Attached Figure Description

[0014] Figure 1 Preparation and characterization of PRP@GEL.

[0015] Figure 2 PRP@GEL has the ability to continuously release growth factors and promotes proliferation.

[0016] Figure 3 PRP@GEL modulates fiberization signals.

[0017] Figure 4 PRP@GEL regulates angiogenesis.

[0018] Figure 5 PRP@GEL promotes endometrial repair in IUA rats.

[0019] Figure 6 PRP@GEL promotes endometrial angiogenesis and anti-fibrosis in IUA rats.

[0020] Figure 7 PRP@GEL can improve the implantation rate and live birth rate in IUA rats.

[0021] Figure 8 Abstract figure: Dual-network hydrogel that targets and activates PRP promotes endometrial repair. Detailed Implementation

[0022] Example 1: Preparation and Characterization of PRP@GEL Hydrogel

[0023] 1) Design and synthesis of HAMA-PBA conjugates:

[0024] 2 g of hyaluronic acid (HA) was dissolved in 10 mL of deionized water (DI) with stirring. Next, 7 mL of methacrylic anhydride (MA) was added dropwise while maintaining the pH of the mixture between 8 and 10. The reaction was allowed to proceed in the dark for 24 hours. Subsequently, the reaction mixture was washed with ethanol, dialyzed against DI water for 5 days, and freeze-dried to obtain methacrylamide hyaluronic acid (HAMA).

[0025] 1 g of HAMA was dissolved in 200 mL of DI water by stirring. Then, 0.5 g of (3-(aminomethyl)phenyl)boronic acid (PBA) and 1.5 g of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride were added to the HAMA solution. The pH of the mixture was maintained at 6.5 and stirred at room temperature for 72 hours. Subsequently, the mixture was dialyzed against deionized water for 5 days and freeze-dried to obtain phenylboronic acid-modified hyaluronic acid methacrylate (HMAM-PBA). Figure 1 A). In Figure 1 In section B, the 1H NMR spectrum shows that HA has been successfully double-modified. Proton signals of 5.71 and 6.09 ppm correspond to the vinyl characteristics of methacrylic acid, while proton signals of 7.45, 7.49, 7.64, and 7.86 ppm correspond to the benzene ring characteristics of phenylboronic acid.

[0026] 2) PRP extraction:

[0027] Blood samples were obtained from 8-week-old healthy SD rats. PRP was extracted using a two-step centrifugation method. 8 mL of whole blood was drawn from each rat under general anesthesia. The sample was then centrifuged at 1000g for 10 minutes, separating into three layers from top to bottom: plasma, leukocyte / platelet layer, and erythrocyte layer. The upper leukocyte / platelet layer and plasma layer were transferred to new centrifuge tubes and centrifuged a second time at 2000g for 10 minutes. The remaining supernatant plasma was discarded, leaving 0.5 mL of plasma containing precipitated platelets. This was thoroughly mixed and identified as PRP. Erythrocytes were carefully removed to minimize interference. The platelet count in the PRP was determined using an automated counter.

[0028] 3) Preparation of PRP@HAMA-PBA-PVA hydrogel

[0029] 6 g of HAMA-PBA, 200 mg of lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP), and 1 g of calcium chloride (CaCl2) were dissolved in 100 mL of PBS (pH 8.5), and the resulting mixture was used as precursor solution A. 1 g of polyvinyl alcohol (PVA) was dissolved in 100 mL of PRP to obtain precursor solution B. Precursor solutions A and B were synthesized into PRP@HAMA-PBA-PVA hydrogel using a dual-tube syringe at a 1:1 volume ratio. The mixture in the custom dual-tube syringe was irradiated under ultraviolet light for 20-30 s. The hydrogel not exposed to ultraviolet light was designated as single-network PRP@HAMA-PBA-PVA, and the hydrogel exposed to ultraviolet light was designated as dual-network PRP@HAMA-PBA-PVA. In subsequent in vitro and in vivo experiments, the HAMA-PBA-PVA hydrogel and the PRP@HAMA-PBA-PVA hydrogel were designated as GEL and PRP@GEL, respectively. Figure 1 C).

[0030] 4) Injection is key to the treatment of IUA. For example... Figure 2 As shown in Figure D, PRP@GEL can be injected effortlessly using a 21G needle without clogging, promoting rapid healing. It is versatile and adaptable to irregular tissue defects. Furthermore, PRP@GEL self-degrades within 14 days. Figure 1 D).

[0031] 5) Figure 1 E shows the rheological properties and self-healing capabilities of the hydrogels, as well as the differences between single-network and dual-network hydrogels. Firstly, when performing oscillatory strain scanning on the uncrosslinked UV-illuminated single-network hydrogel, the storage modulus of the single-network hydrogel was 120 Pa (…). Figure 1 Ei). After subjecting the single-network hydrogel to UV light irradiation in a time-gradient manner, we found that the modulus of the hydrogel could significantly increase with the extension of UV light irradiation time. Figure 1 E-ii). Subsequently, when oscillatory strain scanning was performed on the dual-network hydrogel that had been crosslinked with UV light for 20 s, the storage modulus of the dual-network hydrogel increased to 300 Pa (E-ii). Figure 1 E-iii). Cyclic experiments with shear strain shifting between 1% and 1000% demonstrate that the self-healing properties exhibit good repeatability. Figure 1 E-iv).

[0032] 6) Scanning electron microscopy revealed that the single-network hydrogel exhibited a uniform honeycomb porous structure before UV irradiation. After photocrosslinking, the crosslinking density of the dual-network hydrogel increased, the pore size decreased, and the porous structure became more compact. Figure 1 F).

[0033] Example 2: PRP@GEL exhibits the ability to continuously release growth factors and promote proliferation in vitro.

[0034] 1) PRP contains various growth factors (GFs), including platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and epidermal growth factor (EGF). These GFs have the potential to positively influence the endometrial regeneration process.26,27 However, the sudden release of these GFs after PRP activation limits their application. Here, PRP is loaded into HAMA-PBA-PVA hydrogel via dynamic borate bonding and photocrosslinking. The carboxyl and polyphenol groups provided by HA-PBA chelate Ca... 2+ This method is used to target PRP activation, and the growth factors released by PRP activation can be encapsulated in a 3D hydrogel to further ensure the continuous release of GFs. After immersing the hydrogel in PBS for different times, GFs were detected using an enzyme-linked immunosorbent assay (ELISA). Figure 2 As shown in A, B, and C, PRP experiences explosive GF release within the first 24 hours, followed by balanced release after 72 hours.

[0035] 2) Cell viability after co-culturing with the gel was analyzed using the CCK-8 assay and EdU staining to assess the cytotoxicity of the gel and PRP@GEL. For example... Figure 2 As shown in D and E, no cell proliferation inhibition was observed in any group of HESCs and HUVECs. In HESCs, the absorbance of the PRP@GEL group on day 3 was significantly higher than that of the PRP, GEL, and CTRL groups (p < 0.05). In HUVECs, the absorbance values ​​of both the PRP@GEL and PRP groups on day 3 were higher than those of the CTRL group (p < 0.05). Furthermore, EdU staining of HESCs and HUVECs also showed the same pattern, with more proliferating cells observed in the PRP@GEL group than in the CTRL group (red). Figure 2 FI).

[0036] These results indicate that PRP-containing hydrogels are not cytotoxic to HESCs and HUVECs, but promote cell proliferation.

[0037] Example 3: Modulation of Fibrosis Signals by PRP@GEL

[0038] Endometrial fibrosis is one of the main pathological features of IUA. Therefore, anti-fibrosis is of great significance for the treatment and prevention of IUA33. To investigate the in vitro role of PRP@GEL in endometrial fibrosis, we simulated the potential microenvironmental challenges that HESCs might encounter in the endometrium after treatment with TGF-β1. A TGF-β1-induced in vitro fibrosis model was treated with conditioned medium soaked in GEL, PRP, and PRP@GEL hydrogels. Figure 3 A). The in vitro anti-fibrotic effect was assessed by detecting the expression levels of the corresponding genes. We further explored possible signaling pathways to prevent liver fibrosis. After TGF-β1 stimulation of HESCs, the mRNA expression of fibrosis markers such as COL1A1, α-SMA, and CTGF was significantly upregulated ( Figure 3 (BD) indicates successful induction of the in vitro fibrosis model. GEL treatment alone did not affect the TGF-β1-induced upregulation of COL1A1, α-SMA, and CTGF expression. PRP alone could partially reduce COL1A1 expression, but had no significant effect on α-SMA and CTGF expression. When PRP@GEL treatment was used, the expression of COL1A1, α-SMA, and CTGF was significantly downregulated (BD). Figure 3 BD). Western blot results were consistent. TGF-β1 stimulated the protein expression of COL1A1, α-SMA, and CTGF, while PRP@GEL effectively downregulated the TGF-β1-induced protein expression of COL1A1, α-SMA, and CTGF. Figure 3 EH). Phosphorylation of SMAD2 / 3 is one of the important signaling pathways in fibrosis formation. In the TGF-β1 group, p-SMAD2 / 3 expression was upregulated, and SMAD2 / 3 phosphorylation levels were increased. After PRP@GEL treatment, p-SMAD2 / 3 expression and SMAD2 / 3 phosphorylation levels were significantly reduced. Figure 3 The results showed that PRP@GEL can inactivate the TGF-β1-SMAD2 / 3 pathway in an in vitro fibrosis model.

[0039] In summary, PRP@GEL may regulate HESCs fibrosis by inhibiting the downregulation of fibrosis genes through the TGF-β1-SMAD2 / 3 signaling pathway.

[0040] Example 4: PRP@GEL regulates angiogenesis

[0041] Angiogenesis plays a crucial role in the recovery of intraepithelial neoplasia (IUA), and treatments targeting angiogenesis have proven effective for IUA. Here, we evaluated the regulation of angiogenesis in HUVEC cells. The proliferative effect of PRP@GEL on HUVECs has been previously demonstrated. Wound healing assays showed that the PRP@GEL group achieved 86.65±2.90% healing within 12 hours, compared to 54.69±5.00% in the CTRL group (p<0.001), indicating better cell migration in the PRP@GEL-treated group. Figure 4 A and 5B). Transwell invasion experiments showed that PRP treatment significantly enhanced the invasion of HUVECs, and PRP@GEL treatment further enhanced this effect. Figure 4 (C and 4D). Endothelial cell tube formation experiments revealed the angiogenic effect of PRP@GEL treatment. The number of connections, principal connections, principal segments, number of segments, and segment length were all significantly increased in the PRP@GEL group compared to the CTRL group. Figure 4 EJ). Meanwhile, in the PRP@GEL group, the generally accepted angiogenesis genes VEGFA and PDFGA were increased, while FSTL1 and ANGPT2 showed an increasing trend in the PRP@GEL group, but this was not statistically significant. Figure 4 KN).

[0042] In summary, PRP@GEL can promote angiogenesis, thereby promoting endometrial regeneration and IUA recovery.

[0043] Example 5: Effect of PRP@GEL on endometrial regeneration in IUA rats

[0044] An IUA animal model was established by injecting 95% ethanol into the uterine horn of SD rats to evaluate the in vivo effects of PRP@GEL. Female rats were randomly divided into 5 groups.

[0045] The CTRL group received PBS injections to simulate surgery (sham surgery). IUA models received PBS (ETOH group), GEL (GEL group), PRP (PRP group), and PRP@GEL (PRP@GEL group) treatments for 2 weeks, respectively. Figure 5 A). On postoperative day 14, 14 rats from each group were sacrificed, and gross images and stained tissue sections of the uterus were collected for analysis. Compared with the CTRL group, the ETOH group showed significantly thinner endometrium and fewer glands in the damaged uterine tissue. The PRP@GEL group showed uniform distribution of stroma and epithelium, and increased endometrial thickness and glandular number. Figure 4BG). The average endometrial thickness in the CTRL group was 440.9 μm, with 17.25 glands. In contrast, the endometrial thickness in the ETOH group was only 329.0 μm, with only 2.69 glands. The average endometrial thickness in the PRP@GEL group was 449.3 μm, with an average of 16.38 glands, indicating that PRP@GEL has significant tissue regeneration capacity. Figure 5 F and 5G). Meanwhile, the average uterine thickness and glandular number in the PRP@GEL group were also higher than those in the PRP group (F and 5G). Figure 5 F and 5G) indicate that PRP@GEL has a better effect on promoting endometrial repair.

[0046] We observed the protective effect of PRP@GEL in an in vitro fibrosis model using in vitro cell experiments. To further evaluate its anti-fibrotic effect in rats, we performed Masson's trichrome staining on five groups of rats to detect endometrial collagen deposition. The ETOH group showed a significant increase in endometrial collagen deposition (mean collagen staining 28.88%), significantly higher than the CTRL group (mean collagen staining 7.88%). In contrast, the PRP@GEL group (mean collagen staining 8.04%) showed a 20.84% ​​reduction in collagen deposition compared to the ETOH group and a 12.25% reduction compared to the PRP group. Figure 5 D and 5H). The expression results of endometrial tissue fibrosis markers α-SMA and COL1A1 proteins were consistent. The α-SMA-positive area increased in the ETOH group, while the α-SMA-positive area decreased in the GEL, PRP, and PRP@GEL groups. Figure 6 A and 6B). The positive area of ​​COL1A1 increased in the ETOH group, but decreased only in the PRP@GEL group. Figure 6 (C and 6D). These findings collectively demonstrate the effectiveness of PRP@GEL in alleviating endometrial fibrosis and reducing collagen deposition.

[0047] Since endometrial regeneration relies on angiogenesis to regenerate tissue, our aim was to investigate the PRP@GEL-induced neovascularization observed in vitro. Figure 4 Is there a correlation between ) and endometrial angiogenesis in vivo? Immunohistochemical staining of the rat uterus showed that, compared with the ETOH group, the PRP@GEL group had higher expression of CD31 and vWF ( Figure 6 EH). The above results indicate that PRP@GEL promotes angiogenesis.

[0048] In summary, PRP@GEL can effectively increase endometrial thickness, alleviate endometrial fibrosis, and increase angiogenesis.

[0049] Example 6: PRP@GEL can improve implantation rate and live birth rate in IUA rats.

[0050] Restoring fertility is considered the ultimate goal of endometrial regeneration. The above results demonstrate the effectiveness of PRP@GEL in endometrial regeneration in IUA rats. Whether PRP@GEL can restore fertility in IUA rats requires further investigation.

[0051] This study conducted a comprehensive fertility experiment involving embryo implantation, embryonic development, and live birth. On day 14 post-operation, female SD rats were paired 1:1 with male SD rats, and successful mating was determined by morning vaginal smears. Eight rats from each group were sacrificed 14 days post-coital (dpc), and uteri were collected to analyze the number of implanted embryos and the implantation rate. The number of implantation sites in the ETOH group (1.38±0.89) was significantly lower than that in the CTRL group (12.50±1.12), while the number of implantation sites in the PRP@GEL group (8.38±1.67) was significantly higher than that in the ETOH group, but comparable to that in the CTRL group. Figure 7 A, 7C). Eight female mice in each group continued feeding, and the number of fetuses remained consistent with the experimental results. The number of fetuses in the ETOH group was significantly reduced (1.13±0.79), while PRP@GEL could restore damaged endometrial function. The number of fetuses in the PRP@GEL group (6.88±1.54) was significantly increased compared to the ETOH group, and comparable to the CTRL group (10.63±1.09). Figure 7 B and Figure 7 D). Compared with the CTRL group, the live birth rate in the ETOH group (25%) decreased sharply, while the live birth rate in the PRP@GEL group reached 87.5%. Figure 7 E). There was no statistically significant difference in the average fetal weight among the groups. Figure 7 F). Furthermore, we monitored the development of the offspring in the PRP@GEL group and found that neonatal growth was normal during the 14-12 day period (F). Figure 7 G).

[0052] These results indicate that intrauterine injection of PRP@GEL can significantly increase the number of live births in rats with uterine injury, without adversely affecting the growth and development of offspring.

[0053] In summary, we developed and produced a localized activated PRP (PRA) via injectable dual-network hydrogel (PRP@GEL) through dynamic borate ester bonds and photocrosslinking, and by the conjugation reaction of HAMA-PBA and PVA. Figure 8 A). This special structure and configuration enable PRP to be locally activated within the uterus and to sustainably release growth factors, while simultaneously preventing the cascade release and rapid clearance of active growth factors. Figure 8B). PRP@GEL hydrogel has been shown to possess properties that promote proliferation, angiogenesis, anti-fibrosis, and comprehensive repair of the endometrial microenvironment. Intrauterine perfusion of PRP@HAMA-PBA-PVA can promote endometrial cell proliferation and angiogenesis, reduce fibrosis, thereby restoring endometrial function and promoting embryo implantation. Figure 8 C). Therefore, localized activated PRP prepared via injectable dual-network hydrogels offers a novel and promising approach to the treatment of IUA and has shown potential for tissue regeneration in a variety of applications.

Claims

1. A hydrogel for endometrial repair, characterized in that, It is a hydrogel made from platelet-rich plasma, phenylboronic acid-modified hyaluronic acid methacrylate, lithium phenyl-2,4,6-trimethylbenzoylphosphonate, calcium chloride, and polyvinyl alcohol.

2. The method for preparing a hydrogel for endometrial repair as described in claim 1, characterized in that... Includes the following steps: 1) Dissolve phenylboronic acid-modified hyaluronic acid methacrylate, lithium phenyl-2,4,6-trimethylbenzoylphosphonate, and calcium chloride in PBS solution. Add 5-6 g of phenylboronic acid-modified hyaluronic acid methacrylate, 0.1-0.2 g of lithium phenyl-2,4,6-trimethylbenzoylphosphonate, and 1-2 g of calcium chloride to each 100 mL of PBS solution. The resulting mixed solution is used as precursor solution A. 2) Dissolve polyvinyl alcohol in platelet-rich plasma, add 1-2 g of polyvinyl alcohol to every 100 mL of platelet-rich plasma, and use the resulting polyvinyl alcohol-platelet-rich plasma mixed solution as precursor solution B. 3) Precursor solution A and precursor solution B were mixed in a 1:1 volume ratio to obtain PRP@HAMA-PBA-PVA hydrogel; 4) Place the PRP@HAMA-PBA-PVA hydrogel from step 3) under ultraviolet light for 20-30 seconds to obtain a hydrogel for endometrial repair.

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