Application of injectable bioactive hydrogel in treatment of recurrent abortion

Abstract

The invention provides application of injectable bioactive hydrogel in treatment of recurrent abortion, and relates to the technical field of biomedicine, and the technical key points are as follows: aldehydic hyaluronic acid and chitosan are connected in parallel, the injectable bioactive hydrogel is developed, and the hydrogel is loaded with LIF, VEGF, IL-11 and valproic acid to enhance regeneration activity. According to the application, the effect is evaluated in vitro through a specific verification experiment. Results show that the hydrogel prepared by the application shows rapid gelation, good biocompatibility and factor loading capacity, and enhances the proliferation of human endometrial stromal cells and the vascularization capacity of human umbilical vein endothelial cells. In macrophages, the expression of a proinflammatory cytokine is down-regulated, and the expression of an anti-inflammatory cytokine IL-10 is up-regulated. In conclusion, the HA-CHO / chitosan hydrogel in the application supports endometrial regeneration by synergistically promoting proliferation, angiogenesis, immune regulation and receptivity.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to application of injectable bioactive hydrogel in treating recurrent spontaneous abortion. Background Art

[0002] Recurrent pregnancy loss (RPL), defined as two or more consecutive miscarriages, affects approximately 1-2% of women of reproductive age and remains a troubling clinical problem worldwide. While chromosomal abnormalities, endocrine disorders, and thrombophilia contribute to some cases, a significant proportion of RPL is classified as idiopathic. Emerging evidence suggests that impaired endometrial receptivity and dysfunction of the local immune and vascular microenvironment are major contributing factors. In particular, chronic endometrial damage and inflammation, coupled with insufficient angiogenesis and inadequate stromal remodeling, impair the endometrium's ability to support successful embryo implantation and pregnancy maintenance.

[0003] Current interventions for RPL associated with endometrial insufficiency—such as hormone therapy, platelet-rich plasma (PRP) infusion, or intrauterine granulocyte colony-stimulating factor (G-CSF) administration—have variable efficacy and lack sustained efficacy. Furthermore, these strategies often fail to address the complex interplay between epithelial integrity, vascular remodeling, immune regulation, and receptivity-related signaling. Therefore, there is a pressing need for biomaterial-based solutions that can locally restore endometrial structure and function by combining mechanical support with biological manipulation. Summary of the Invention

[0004] The purpose of the present invention is to solve the problem in the prior art of lacking biomaterials for local restoration of endometrium.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions: An injectable bioactive hydrogel is characterized in that: the hydrogel is prepared by cross-linking aldehyde-modified hyaluronic acid and chitosan, and the hydrogel is loaded with at least one of leukemia inhibitory factor, vascular endothelial growth factor, interleukin-11 and valproic acid.

[0006] Preferably, the preparation method of the hydrogel is as follows: S1: Synthesis of aldehyde-modified hyaluronic acid; S2: chitosan solution synthesis; S3: Hydrogel formation and bioactive factor loading: The aldehyde-modified hyaluronic acid in S1 is mixed with the chitosan solution in S2, and a bioactive factor is added during the mixing process, wherein the bioactive factor is at least one of leukemia inhibitory factor, vascular endothelial growth factor, interleukin-11 and valproic acid; the mixing time is 2-3 minutes.

[0007] Preferably, the aldehyde-hydrated hyaluronic acid used in S3 is a aldehyde-hydrated hyaluronic acid solution with a concentration of 2% w / v, the chitosan solution is a 2% w / v chitosan solution, and the volume ratio of the aldehyde-hydrated hyaluronic acid solution to the chitosan solution in S3 is 1:1.

[0008] Preferably, the synthesis method of the aldehyde-modified hyaluronic acid in S1 is as follows: Hyaluronic acid was oxidized with sodium periodate (NaIO4, 5 mM) at room temperature in the dark for 4 h, followed by dialysis and freeze-drying.

[0009] Preferably, the chitosan solution in S2 is formed by dissolving chitosan in 0.5% acetic acid.

[0010] The present application also provides an injectable bioactive hydrogel for use in preparing a drug for treating recurrent spontaneous abortion. The injectable bioactive hydrogel is the injectable bioactive hydrogel described above.

[0011] Preferably, the medicament is used to enhance endometrial regeneration to treat recurrent miscarriage.

[0012] Preferably, the drug enhances endometrial regeneration through angiogenesis and immunomodulation to treat recurrent miscarriage.

[0013] Compared with the prior art, this application has the following beneficial effects: This application developed an injectable bioactive hydrogel by combining aldehyde-modified hyaluronic acid with chitosan. This hydrogel is loaded with LIF (leukemia inhibitory factor), VEGF (vascular endothelial growth factor), IL-11 (interleukin-11), and valproic acid to enhance regenerative activity. The hydrogel's efficacy was evaluated in vitro using cell proliferation (CCK-8 assay), angiogenesis (HUVEC assay), cytokine expression (THP-1 cell qPCR), and endometrial gene profiling (hEMSC cell qPCR). The results demonstrated that the hydrogel exhibited rapid gelation, good biocompatibility, and factor-loading capacity. It significantly enhanced the proliferation of human endometrial stromal cells (hEMSC) and the angiogenesis of human umbilical vein endothelial cells (HUVEC). In macrophages, the expression of proinflammatory cytokines (TNF-α, IL-6) was downregulated, while the expression of the anti-inflammatory cytokine IL-10 was upregulated. The hydrogel also increased the expression of LIF and IGFBP1 (insulin-like growth factor binding protein 1), but not PRL (prolactin), suggesting that it enhanced endometrial receptivity without leading to complete decidualization. In summary, the HA-CHO / chitosan hydrogel in this application supports endometrial regeneration by synergistically promoting proliferation, angiogenesis, immunomodulation, and receptivity. It has significant potential for the local treatment of RPL associated with endometrial insufficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Schematic diagram of the preparation of an injectable bioactive hydrogel based on aldehyde-modified hyaluronic acid (HA-CHO) and chitosan in one embodiment of the present invention.

[0015] Figure 2 In one embodiment of the present invention, CCK-8 assay showed that endometrial stromal cells cultured with control medium and hydrogel extracts proliferated over time.

[0016] Figure 3 Figure 2 shows the effect of a hydrogel extract on in vitro endothelial tube formation according to one embodiment of the present invention. (A) HUVECs cultured in standard culture medium (control) exhibit sparse, poorly branched tubular structures. (B) In contrast, cells cultured in the hydrogel extract formed a dense, interconnected capillary network with more branch points and elongated tubes. Scale bar: 100 μm.

[0017] Figure 4Figure 1 shows a quantitative analysis of tube formation in one embodiment of the present invention: total tube length and node number in HUVEC networks under different conditions. HUVECs were cultured on Matrigel with either standard culture medium (control) or hydrogel extract. After 8 hours, total tube length (left Y-axis, orange bars) and node number (right Y-axis, green bars) were quantified using ImageJ. Hydrogel extract significantly enhanced both vascular parameters.

[0018] Figure 5 This study demonstrates that a hydrogel extract, according to one embodiment of the present invention, modulates inflammatory cytokine expression in THP-1-derived macrophages. THP-1-derived macrophages were stimulated with either the hydrogel extract or standard culture medium for 24 hours. Gene expression of pro-inflammatory markers (TNF-α and IL-6) and anti-inflammatory cytokines (IL-10) was analyzed by qPCR. Values ​​were normalized to those of the control group. The hydrogel extract significantly inhibited TNF-α and IL-6 expression while upregulating IL-10.

[0019] Figure 6 This study demonstrates that a hydrogel extract, according to one embodiment of the present invention, promotes the expression of endometrial receptivity-related genes in human endometrial stromal cells (hEMSCs). hEMSCs were cultured for 48 hours in either control medium or the hydrogel extract. The expression of PRL, IGFBP1, and LIF was measured by qPCR and normalized to the control. The hydrogel extract significantly enhanced LIF expression and modestly increased IGFBP1 expression, while simultaneously reducing PRL levels. DETAILED DESCRIPTION

[0020] The present invention is further described in detail below with reference to specific embodiments.

[0021] The present application provides an injectable bioactive hydrogel, which is prepared by cross-linking aldehyde-modified hyaluronic acid (HA-CHO) with chitosan.

[0022] The preparation method of the injectable bioactive hydrogel is as follows: S1: Synthesis of aldehyde-modified hyaluronic acid (HA-CHO): Hyaluronic acid was oxidized with sodium periodate (NaIO4, 5 mM) at room temperature in the dark for 4 h, followed by dialysis and freeze-drying. The specific steps for the synthesis of aldehyde-modified hyaluronic acid (HA-CHO) are as follows: HA is oxidized with sodium periodate (NaIO4, 5 mM) for 4 hours at room temperature in the dark. The oxidized hyaluronic acid solution is then placed in a dialysis bag with a molecular weight cutoff of 3.5 kDa and dialyzed against 0.1 M phosphate buffer (pH 7.4) at 4°C for 24-48 hours, with the dialysis solution replaced every 6-8 hours. After dialysis, the solution is placed in a pre-freezing stage at -40°C for 2-4 hours to completely freeze the solution. The solution is then sublimated and dried under a vacuum degree of less than 0.133 kPa at around -25°C for 12-24 hours. Finally, the solution is subjected to desorption drying at 25°C for 6-12 hours to obtain aldehyde-modified hyaluronic acid powder.

[0023] S2: Chitosan solution preparation: Chitosan was dissolved in 0.5% acetic acid to prepare a 2% (w / v) solution.

[0024] S3: Hydrogel formation: Under sterile conditions, equal volumes of HA-CHO solution (2% w / v) and chitosan solution were mixed. Bioactive factors (LIF 100 ng / mL, VEGF 50 ng / mL, IL-11 100 ng / mL, VPA 0.5 mM) were added during the mixing process to achieve the purpose of loading the hydrogel with bioactive factors. The aldehyde-modified hyaluronic acid solution (2% w / v) used was prepared by dissolving synthetically obtained aldehyde-modified hyaluronic acid powder in an appropriate amount of sterile water. The chitosan solution was a 2% w / v chitosan solution prepared by dissolving chitosan in 0.5% acetic acid. Equal volumes of the two solutions were mixed, and the bioactive factors were added during the mixing process. The mixing time was 2-3 minutes to ensure sufficient hydrogel formation and uniform loading of the bioactive factors. The concentrations of the bioactive factors added here refer to their final concentrations in the hydrogel, for example, 100 ng / mL for LIF, 50 ng / mL for VEGF, 100 ng / mL for IL-11, and 0.5 mM for VPA.

[0025] like Figure 1 As shown, the hydrogel gelated within 2–3 min by forming a Schiff base ( Figure 1 ).

[0026] like Figure 1 As shown, the injectable hydrogel was prepared by mixing aldehyde-functionalized hyaluronic acid (HA-CHO) with chitosan in acetic acid. A Schiff base reaction between the aldehyde and amino groups resulted in rapid gelation under mild aqueous conditions, forming a stable and biocompatible network. Notably, the hydrogel system was designed to allow for the incorporation of bioactive agents during the mixing process.

[0027] Specifically, this application utilizes the hydrogel to encapsulate key bioactive factors associated with endometrial repair, including leukemia inhibitory factor (LIF) and vascular endothelial growth factor (VEGF) for promoting endometrial receptivity and angiogenesis, as well as interleukin-11 (IL-11) and valproic acid (VPA) for supporting epithelial regeneration and epigenetic regulation. These factors are physically embedded or retained within the network through electrostatic interactions during the gelation process. Therefore, this platform provides a versatile delivery vehicle for the sustained local release of regenerative signals, laying the foundation for subsequent in vitro studies.

[0028] like Figure 1 As shown, Figure 1 The concept and chemical basis of the hydrogel formulation used in this application are presented in this application, in which low immunogenicity and potential for intrauterine application are ensured by the use of biocompatible polymers - HA and chitosan. The dynamic Schiff base linkages formed between the aldehyde groups on HA-CHO and the amino groups on chitosan not only facilitate the rapid gelation of the system but also make it injectable, which is crucial for minimally invasive delivery. Importantly, this reversible covalent bond also gives the hydrogel mild self-healing potential and responsiveness to physiological environments (such as pH changes). In addition, the modular nature of the system allows the incorporation of therapeutic peptides or growth factors, providing versatility for endometrial repair. Figure 1 As shown, the hydrogel preparation is the basic material for subsequent in vitro biological validation experiments.

[0029] The above contents are described below with reference to specific embodiments: Experimental materials and sources: Hyaluronic acid (HA, molecular weight ~200 kDa), chitosan (medium molecular weight, deacetylation degree ≥85%), sodium periodate, acetic acid, leukemia inhibitory factor (LIF), vascular endothelial growth factor (VEGF), interleukin-11 (IL-11), valproic acid (VPA), and all cell culture reagents were purchased from Sigma-Aldrich (unless otherwise specified).

[0030] Human endometrial stromal cells (hEMSCs) and human umbilical vein endothelial cells (HUVECs) were purchased from ScienCell, and Matrigel was purchased from Corning.

[0031] Example 1: Cell proliferation assay (CCK-8) hEMSCs were divided into two groups and seeded in 96-well plates at a density of 5000 cells / well.

[0032] After 24 h, the original culture medium of the two groups of hEMSCs was replaced with fresh control culture medium and hydrogel extract (prepared by incubating hydrogels in serum-free culture medium at 37°C for 24 h), respectively.

[0033] At 0, 24, 48, 72, and 96 hours, cell viability was measured using a CCK-8 kit (Dojindo, Japan) according to the manufacturer's instructions. The absorbance was recorded at a wavelength of 450 nm using a microplate reader ( Figure 2 ).

[0034] See also Figure 2 The OD450 values ​​of endometrial stromal cells co-cultured with the hydrogel extract consistently increased compared to the control group at all time points. At 48 hours, the OD450 value of the hydrogel group reached 0.72, compared to 0.58 in the control group; this difference further increased at 72 hours (1.02 vs. 0.82) and 96 hours (1.21 vs. 0.97). These results suggest that the hydrogel extract provides a favorable microenvironment for cell proliferation, possibly due to the release of bioactive components or improved cell-substrate interactions.

[0035] Figure 2 The proliferation profile shown confirms the cytocompatibility and bioactivity of the HA-CHO / chitosan-based hydrogel. The observed enhanced cell growth over time suggests that soluble factors released from the hydrogel may stimulate the metabolic activity and proliferation of endometrial stromal cells. This could be attributed to residual aminopolysaccharides retained in the hydrogel during the preparation process or to incorporated bioactive signaling molecules. Importantly, the hydrogel not only maintained a nontoxic environment but also actively promoted cell expansion, highlighting its potential utility in endometrial regeneration strategies. These findings support the role of the hydrogel as a scaffold that may aid in epithelial repair and restoration of endometrial thickness in patients with recurrent miscarriage.

[0036] Example 2: Angiogenesis Experiment Matrigel (50 µL / well) was added to a pre-chilled 96-well plate and polymerized at 37°C for 30 minutes.

[0037] HUVECs were divided into two groups and suspended in control culture medium and hydrogel extract, respectively, at a concentration of 2 × 10 4 Cells were seeded on Matrigel at a density of 10 cells / well. After 8 hours of incubation, phase contrast images were acquired using an inverted microscope. Total tube length and node number were quantitatively analyzed using ImageJ software and its "Angiogenesis Analyzer" plug-in ( Figure 3 and Figure 4 ).

[0038] Figure 3 Representative phase contrast images of HUVECs cultured on Matrigel for 8 h under two different conditions are shown. Figure 3 In the A), cells formed relatively simple and sparse capillary-like structures with limited branches and short tube lengths. In contrast, the hydrogel extract group ( Figure 3 B) Shows significantly enhanced tube-forming ability, characterized by increased network density, more junctions (nodes), and longer continuous tubes. Quantitative analysis of total tube length and node number (not shown) confirmed that the hydrogel extract group was superior to the control group in promoting angiogenic behavior.

[0039] Figure 3 The enhanced tube-forming capacity demonstrated by the HA-CHO / chitosan hydrogel extract stimulated the angiogenic activity of endothelial cells in vitro. This angiogenic effect may be attributed to the release of bioactive polysaccharides from the hydrogel matrix or the incorporation of signaling molecules. The presence of dynamic imine bonds and polysaccharide degradation products may also create a microenvironment that is favorable for vascular morphogenesis. This is particularly important for intrauterine applications, as revascularization of endometrial tissue is crucial for embryo implantation and regeneration. In summary, Figure 3 The developed hydrogel not only supports the proliferation of endometrial stromal cells ( Figure 2 ), also promotes the hypothesis of vascular network formation, providing a synergistic strategy for endometrial repair in the setting of recurrent miscarriage.

[0040] Figure 4 The quantitative data in the study showed that the total tube length and number of nodes in the hydrogel extract group were significantly increased compared with the control group. Specifically, the total tube length in the hydrogel extract group reached approximately 3850 μm, while that in the control group was 2400 μm. Similarly, the number of nodes increased from 22 in the control group to 38 in the hydrogel extract group. These indicators clearly indicate that the soluble components released by the hydrogel preparation have a pro-angiogenic effect. This result is consistent with the results of the literature review. Figure 3 The morphological findings were consistent with those of the controls and provided quantitative evidence for an enhanced angiogenic response.

[0041] Figure 4The quantified enhanced endothelial tube formation capacity further supports the bioactivity of the hydrogel system. This angiogenesis-stimulating effect is likely driven by pro-regenerative and pro-angiogenic factors such as VEGF, Figure 1 As shown, VEGF has been incorporated into the hydrogel. In addition, LIF can synergize with VEGF to promote endothelial cell migration and network stabilization, while IL-11 may indirectly support angiogenesis by regulating the inflammatory microenvironment.

[0042] The combined action of these factors resulted in the formation of a significantly more developed and interconnected vascular network in vitro. This is particularly relevant to endometrial repair, as angiogenesis plays a key role in restoring endometrial receptivity and nutrient delivery. Figure 4 This provides functional validation of the hydrogel's potential to support vascular remodeling in the treatment of recurrent miscarriage.

[0043] Example 3: Inflammatory cytokine expression (qPCR analysis of THP-1 macrophages) THP-1 monocytes were induced to differentiate into macrophages using 100 nM PMA for 48 hours.

[0044] Subsequently, the macrophages were divided into two groups and treated with control culture medium and hydrogel extract for 24 hours, respectively. Total RNA was extracted using TRIzol reagent and reverse transcribed using the PrimeScript RT kit. SYBR Green qPCR (ABI 7500 system) was used to detect the gene expression of TNF-α, IL-6, and IL-10, with GAPDH as the internal reference gene for normalization ( Figure 5 ).

[0045] like Figure 5 As shown, exposure to the hydrogel extract significantly altered the cytokine expression profile of THP-1-derived macrophages. Compared with the control group, the mRNA levels of TNF-α and IL-6 were suppressed to 0.45-fold and 0.62-fold, respectively, indicating an anti-inflammatory effect. In contrast, the anti-inflammatory cytokine IL-10 was strongly induced, reaching 2.1-fold that of the control group. These results indicate that the hydrogel extract effectively shifts the macrophage phenotype toward a regulatory or M2-like phenotype.

[0046] Figure 5 The immunomodulatory activity shown suggests that HA-CHO / chitosan hydrogels not only support endometrial tissue regeneration but also contribute to the creation of a favorable immune microenvironment. The reduction in proinflammatory markers (TNF-α, IL-6) and the significant increase in IL-10 may be influenced by factors incorporated into the hydrogel formulation (e.g., IL-11 and VPA, see Figure 1These molecules are known to reprogram the activation state of macrophages and suppress inflammation, which is particularly relevant in recurrent miscarriage, as excessive endometrial inflammation is a pathogenic factor. The results demonstrate that the hydrogel works through both structural and immunomodulatory mechanisms to support endometrial healing.

[0047] Example 4: Endometrial stromal cell gene expression hEMSCs were divided into two groups and cultured at 2 × 10 5 Cells were seeded at a density of 10 cells / well in 6-well plates.

[0048] After 24 hours, the original culture medium was replaced with control culture medium and hydrogel extract in the two groups, respectively.

[0049] After 48 hours of incubation, total RNA was extracted and the gene expression of PRL, IGFBP1, and LIF was analyzed by qPCR. The expression levels were normalized to GAPDH and expressed as fold change relative to the control group ( Figure 6 ).

[0050] exist Figure 6 In the study, qPCR analysis revealed distinct transcriptional responses in endometrial stromal cells treated with the hydrogel extract. Compared with the control group, LIF expression was upregulated by more than threefold, indicating a significant enhancement of endometrial receptivity signaling. IGFBP1 expression was also modestly increased by approximately 1.9-fold, while the decidualization marker PRL was slightly downregulated. These results suggest that the hydrogel selectively activated gene pathways associated with endometrial receptivity without inducing complete decidualization.

[0051] Figure 6 The gene expression changes shown support the ability of the hydrogel to modulate the phenotype of endometrial stromal cells toward a pro-receptive state. A dramatic increase in the key implantation cytokine, LIF, was associated with its incorporation into the hydrogel formulation (see Figure 1 ) and highlights the potential of this material to improve uterine receptivity. Upregulation of IGFBP1 further suggests partial activation of stromal maturation pathways. The slight decrease in PRL may reflect a regulatory balance that favors tissue receptivity without inducing premature decidualization, which could be deleterious during the early implantation phase.

[0052] Statistical analysis Unless otherwise noted, all experiments were performed in triplicate. Data are presented as mean ± standard deviation (SD). Statistical significance was determined using an unpaired, two-tailed Student's t-test or one-way analysis of variance (ANOVA) with Tukey's post hoc test, as appropriate. A p value of < 0.05 was considered statistically significant.

[0053] Based on the above verification experiments, this application confirms that the hydrogel described above provides dual biochemical and microenvironmental signals, shifting the gene expression pattern of hEMSCs to a mode that supports embryo implantation, verifying its therapeutic relevance in endometrial repair for recurrent miscarriage.

[0054] Based on this, this application developed a bioactive injectable hydrogel based on aldehyde-modified hyaluronic acid (HA-CHO) and chitosan, designed for endometrial regeneration in patients with recurrent miscarriage. The hydrogel was prepared by mild Schiff base cross-linking and exhibited excellent structural properties, injectability, and the ability to load and release regenerative factors such as LIF, VEGF, IL-11, and VPA. Figure 1 In vitro experiments confirmed the good biocompatibility of the hydrogel and its ability to continuously promote the proliferation of human endometrial stromal cells (hEMSCs). Figure 2 Furthermore, the hydrogel extract enhanced the formation of capillary-like tubular structures and node development ( Figure 3 and Figure 4 ), significantly promoted the angiogenesis of HUVECs, which is likely due to the release of VEGF. Importantly, the hydrogel also has an immunomodulatory effect: it inhibits pro-inflammatory cytokines (TNF-α, IL-6) while significantly upregulating IL-10, indicating that it can promote the transformation of the microenvironment to an anti-inflammatory state ( Figure 5 Gene expression analysis further showed that the hydrogel could upregulate key markers of endometrial receptivity (especially LIF and IGFBP1), but did not lead to increased PRL expression, indicating that it can prevent excessive decidualization ( Figure 6 This property suggests that the hydrogel can target and activate pathways that support regeneration and embryo implantation.

[0055] In summary, HA-CHO / chitosan hydrogel serves as a multifunctional platform capable of supporting cell proliferation, promoting angiogenesis, modulating inflammatory responses, and enhancing endometrial receptivity. This study lays an important foundation for future translational therapeutic strategies for uterine cavity injury and recurrent miscarriage.

Claims

1. An injectable bioactive hydrogel, characterized in that: The hydrogel is prepared by cross-linking aldehyded hyaluronic acid and chitosan, and at least one of leukemia inhibitory factor, vascular endothelial growth factor, interleukin-11 and valproic acid is loaded into the hydrogel.

2. The injectable bioactive hydrogel according to claim 1, characterized in that: The preparation method of the hydrogel is as follows: S1: Synthesis of aldehyde-modified hyaluronic acid; S2: chitosan solution synthesis; S3: Hydrogel formation and bioactive factor loading: The aldehyde-modified hyaluronic acid in S1 is mixed with the chitosan solution in S2, and a bioactive factor is added during the mixing process, wherein the bioactive factor is at least one of leukemia inhibitory factor, vascular endothelial growth factor, interleukin-11 and valproic acid; the mixing time is 2-3 minutes.

3. The injectable bioactive hydrogel according to claim 2, wherein: The aldehyde-hydrated hyaluronic acid used in S3 is a aldehyde-hydrated hyaluronic acid solution with a concentration of 2% w / v, and the chitosan solution is a 2% w / v chitosan solution. The volume ratio of the aldehyde-hydrated hyaluronic acid solution to the chitosan solution in S3 is 1:

1.

4. An injectable bioactive hydrogel according to claim 2, characterized in that: The synthesis method of the aldehyde-modified hyaluronic acid in S1 is as follows: Hyaluronic acid was oxidized with sodium periodate (NaIO4, 5 mM) at room temperature in the dark for 4 h, followed by dialysis and freeze-drying.

5. The injectable bioactive hydrogel according to claim 2, wherein: The chitosan solution in S2 is formed by dissolving chitosan in 0.5% acetic acid.

6. Use of an injectable bioactive hydrogel in the preparation of a drug for treating recurrent spontaneous abortion, characterized in that: The injectable bioactive hydrogel is the injectable bioactive hydrogel according to any one of claims 1 to 5.

7. Use of an injectable bioactive hydrogel according to claim 6 in the preparation of a drug for treating recurrent spontaneous abortion, characterized in that: The drug is used to enhance endometrial regeneration to treat recurrent miscarriage.

8. Use of an injectable bioactive hydrogel according to claim 7 in the preparation of a drug for treating recurrent spontaneous abortion, characterized in that: The drug enhances endometrial regeneration through angiogenesis and immunomodulation to treat recurrent miscarriage.

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