English

• Endometrial injury significantly affects women's reproductive health, often arising from inadequate intrauterine interventions, such as repeated abortions or frequent curettage within short intervals [1,2]. Intrauterine adhesion (IUA) occurs when endometrial healing is impaired, leading to partial or complete blockage of the uterine cavity that can cause infertility and recurrent miscarriage [3,4]. The standard treatment for IUA is hysteroscopic adhesiolysis, which is a procedure used to remove adhesions and restore uterine anatomy [5]. However, despite advancements in surgical inadequate, recurrence rates remain high, and endometrial restoration is often inadequate, negatively affecting reproductive outcomes [6,7]. Adjunct therapies, such as estrogen administration [8] and intrauterine balloons [9,10] have demonstrated limited efficacy in preventing re-adhesion and facilitating tissue regeneration. Therefore, there is an urgent need for novel, effective, and targeted treatments for IUA.

  Endometrial receptivity—crucial for successful blastocyst implantation—is modulated by various cytokines [11,12]. It has been reported that endometrial receptivity is notably diminished after endometrial injury [13]. Studies have indicated that vascularization is a major component of endometrial receptivity, closely associated with levels of insulin-like growth factor 1 (IGF-1) [14,15]. Besides, IGF-1 plays a crucial role in cellular behavior and facilitates endometrial regeneration [16-18]. In summary, IGF-1 is a significant factor that affects fertility [19]. However, several factors limit its practical applications. IGF-1 has a short biological half-life that necessitates frequent dosing to maintain therapeutic levels [20]. It is expensive, sensitive to environmental conditions, and degrades unless specific storage and transport conditions are maintained. These challenges hinder the use of IGF-1 as a treatment for endometrial injury, underscoring the need for further research to develop practical and effective IGF-1 delivery strategies for IUA treatment.

  Hydrogels, with their unique physicochemical properties, have emerged as promising candidates in clinic [21-25]. Specifically, hydrogels that possess injectable and self-healing capabilities offer significant advantages in the prevention and repair of IUA. The injectability of hydrogels allows for minimally invasive administration directly to the site of uterine injury. Moreover, the self-healing property of hydrogels enhances their durability and resilience, ensuring that the barrier remains effective even when subjected to mechanical stresses inherent to the uterine environment. This property is critical for maintaining continuous protection of the injured tissue surfaces. Self-assembled peptide-derived supramolecular hydrogels have emerged as a promising approach. Self-assembled peptides offer multiple non-covalent interactions, including hydrogen bonding, hydrophobic effects, and electrostatic interactions, enabling them to interact with bioactive proteins and co-assemble into ordered nanostructures, such as nanofibers, resulting in hydrogel formation [26-29]. Peptide-based hydrogels are relatively simple, cost-effective, and inherently biodegradable, decomposing into amino acids through natural metabolic processes thus alleviating concerns regarding long-term accumulation and potential toxicity [30-33]. Their exceptional biocompatibility minimizes the risks of immune rejection and adverse reactions [34,35]. Additionally, the bioactivity of these hydrogels can be modified by incorporating specific sequences and functionalized chemical groups during peptide design. This customization facilitates the development of hydrogels that address the specific needs of various therapeutic applications, thereby enhancing their effectiveness [36-38]. Recent research in endometrial repair focuses on the implantation of anti-adhesion materials with antifibrotic and anti-inflammatory agents, stem cells, or bioactive components to enhance therapeutic effects [39-41]. For example, Xin et al. developed a hyaluronic acid gel loaded with stem cells to prevent adhesion and facilitate cellular activity in the damaged areas [42]. However, drug release challenges inherent to hydrogel structures can result in suboptimal therapeutic outcomes, and synthetic processes may inactivate bioactive agents.

  Therefore, we developed a two-component peptide-based hydrogel that self-assembled from a biotinylated peptide to load an IGF-1 mimetic peptide (IGFp), known for its self-assembly capabilities and IGF-1 bioactivity. The resulting hydrogel exhibits excellent mechanical properties and retained native IGF-1 bioactivity. Our findings revealed that the two-component hydrogel significantly enhanced endometrial functional recovery by enhancing tissue regeneration, angiogenesis, and endometrial receptivity. Consequently, it holds promise as a potential treatment for endometrial injuries and preventing intrauterine adhesion formation.

  To explore the use of peptide-based hydrogels for treating endometrial injury and preventing intrauterine adhesion occurrence. An efficient self-assembled biotinylated peptide hydrogel Biotin-^DFYIGSRGD (Biotin-pep, Fig. 1A), with significant adhesion and mechanical properties, has been reported for 3D cell culture and effective cell spheroid production [43,44]. Meanwhile, an IGFp composed of a hydrophobic self-assembly motif (Nap-FF) and the C-region of IGF-1 (GYGSSSRRAPQT) has been shown to mimic the bioactivity of native IGF-1 [45]. We believe that the non-covalent bond between IGFp and biotin-pep would enhance the mechanical strength of the two-component peptide hydrogel (biotin-pep/IGFp) and be beneficial for the treatment of endometrial injury.

  Figure 1

  

  Figure 1.  Design and characterization of the two-component peptide hydrogel (A) Chemical structure of Biotin-pep. (B, C) Optical images of Biotin-pep hydrogel before and after adding IGFp. (D, E) TEM morphology of Biotin-pep hydrogel before and after adding IGFp. (F) Dynamic frequency sweep of Biotin-pep hydrogel before and after adding IGFp. (G) Fourier transforms infrared spectroscopy Biotin-pep hydrogel before and after adding IGFp. (H) Detection of cycling breaking and recovery ability of Biotin-pep/IGFp hydrogel.

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  Biotin-pep and IGFp were obtained through standard Fmoc-based solid-phase peptide synthesis, purified by reverse-phase high-performance liquid chromatography, and verified by liquid chromatography-mass spectrometry (Figs. S1 and S3 in Supporting information showed that the cationic peak signals of Biotin-pep and IGFp were 1140.3 and 1786.6, respectively, consistent with their molecular weights, indicating successful synthesis of the two peptides) and nuclear magnetic resonance (NMR) (Fig. S2 in Supporting information). Biotin-pep formed a semi-transparent hydrogel in phosphate-buffered saline (pH 7.4) at a concentration of 0.8 wt% through a heating-cooling treatment (Fig. 1B). Transmission electron microscopy (TEM) revealed that biotin-pep formed long nanofibers (diameter of ~8 nm) in the hydrogel (Fig. 1D). Upon IGFp addition, biotin-pep/IGFp formed an opaque hydrogel (Fig. 1C), and TEM revealed the presence of nanosheets with diameters of 20–120 nm (Fig. 1E). In women of childbearing age, the endometrial glands secrete large amounts of endometrial mucus daily, causing the injected hydrogel to be rapidly expelled from the uterine cavity. Hydrogels with good viscoelasticity can hinder the rapid clearance of secreted mucus. Therefore, we studied the rheological properties of biotin-pep hydrogels before and after IGFp addition. The elastic modulus (G') of the Biotin-pep hydrogel was higher than of the viscosity modulus (G''), and the frequency dependence between 0.1 and 100 rad/s was weak, indicating the formation of a stable hydrogel. The G' of the Biotin-pep hydrogel was approximately 765 Pa, which increased to about 1611 Pa following IGFp addition (Fig. 1F and Fig. S4 in Supporting information).

  This enhancement may be because of the enhanced non-covalent interactions (hydrogen bonds and hydrophobic interactions) caused by the co-assembly of biotin-pep and IGFp. In vitro degradation experiment, this hydrogel gradually degraded over a period of 6 days in stimulated uterine fluid (Fig. S5 in Supporting information). Therefore, the resultant two-component peptide hydrogel exhibited significant mechanical properties and stability, matching the uterine cavity wall and avoiding the rapid clearance of physiological mucus secretion. Additionally, Fourier transform infrared spectroscopy demonstrated that the peak in the amide I region (1636 cm⁻¹, C=O stretching vibration) indicated the presence of a β-sheet conformation in the biotin-pep hydrogel (Fig. 1G). When IGFp participated in the self-assembly of biotin-pep, the amide I region peak was clearly enhanced, indicating the formation of a highly ordered nanostructure in the two-component peptide hydrogel. Notably, the biotin-pep/IGFp hydrogel exhibited shear-induced phase transition and self-healing properties. The hydrogel transforms into a solution by simple vortexing and reverts to a hydrogel after approximately 30 min, which is crucial for its injectability (Fig. 1H).

  In clinical practice, IUA is primarily caused by mechanical damage from procedures, such as abortion and curettage. Thus, we established an endometrial injury rat model based on mechanical damage, following previously reported methods [46]. All experimental procedures were conducted in compliance with the National Research Council's "Guide for the Care and Use of Laboratory Animals" and received approval from the Experimental Animal Center of the First Affiliated Hospital of Wenzhou Medical University (No. WYYY-IACUC-AEC-2024–053). To minimize variability among the rats, a self-control model was employed, where the left uterus remained untreated, and the right uterus was mechanically scraped to replicate the adhesion-inducing conditions (Fig. 2A, Figs. S6 and S7 in Supporting information). This approach ensured that any observed differences can be attributed to mechanical interventions.

  Figure 2

  

  Figure 2.  Biotin-pep/IGFp hydrogel promotes the regeneration of injured endometrium in vivo experiment. (A) Schematic diagram of animal study design. (B) Representative H & E-stained images of endometrium for each group. (C) Representative Masson-stained images of endometrium for each group (scale bar: upper image, 500 µm; lower image, 200 µm). (D–F) Relative gland number (D), relative thickness (E), and relative fibrosis rate (F) of injured endometrium compared to self-controls in each group. Results are presented as mean ± SEM (n = 4 per group). P < 0.05, **P < 0.01. n.s., no significance.

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  To assess the therapeutic potential of hydrogels for endometrial repair, we first co-incubated the hydrogel with endometrial stromal cells in vitro and observed favorable biocompatibility (Figs. S8 and S9 in Supporting information). In vivo, hematoxylin and eosin staining was used to observe morphological characteristics among the various treatment groups (Fig. 2B). The control side exhibited a complete columnar epithelium and numerous round glands embedded within the stroma, indicating a healthy endometrium. In contrast, the damaged endometrial surfaces were often disorganized or absent in certain regions, with a significant reduction in glandular structures following injury. Treatment with biotin-pep/IGFp hydrogel markedly increased the number of glands and moderately enhanced endometrial thickness, thereby facilitating the morphological recovery of damaged tissues (Figs. 2D and E). Endometrial fibrosis, a key histological feature of impaired endometrial repair, is often associated with the formation of a non-functional endometrium. Therefore, we used Masson's staining to assess collagen deposition in the endometrium of each group (Fig. 2C). The results demonstrated substantial collagen accumulation in the stromal layer of the damaged endometrium compared to that in the control side. Notably, biotin-pep/IGFp hydrogel treatment significantly reduced collagen deposition, indicating an antifibrotic effect (Fig. 2F). Moreover, the application of biotin-pep without IGFp showed a non-significant trend of improvement post-injury, as indicated by both general observation and Masson's trichrome staining. In addition, the expression of transforming growth factor beta 1 (TGF-β1) level was increased in the injury group, while it was decreased in biotin-pep/IGFp hydrogel group (Fig. S10 in Supporting information).

  Endometrial receptivity is crucial during the implantation window for blastocyst implantation and is significantly affected by various cytokines. It is essential for the endometrial reproductive functions. Imbalanced cytokine profiles or disrupted hormonal signaling can lead to implantation failure, highlighting the significance of harmonious interactions among these factors. Damage to the endometrial basal layer can alter the expression of estrogen and progesterone receptors (ER and PR, respectively), consequently affecting hormone responsiveness. Therefore, assessing the expression of these receptors was crucial to determine the endometrial receptivity. Our study revealed that PR expression was significantly reduced in damaged tissues, whereas ER expression was relatively unchanged compared with that in the normal tissues (Figs. 3A and B). This alteration disrupts endometrial function, affects the regeneration and shedding cycle, and potentially impairs the implantation potential and fertility. PR expression was restored following biotin-pep/IGFp hydrogel treatment (Figs. 3D and E).

  Figure 3

  

  Figure 3.  Biotin-pep/IGFp hydrogel improve endometrial receptivity in vivo experiment of injured endometrium. (A, B) Representative immunohistochemical staining of progesterone receptors (A) and estrogen receptors (B) in injured endometrium among different treatment groups. scale bar: upper image, 100 µm; lower image, 50 µm. (C) Relative quantity of blood vessels in damaged endometrium of different treatment groups (blue: 2-(4-amidinophenyl)−6-indolecarbamidine dihydrochloride (DAPI); green: CD31; red: α-SMA. Scale bar: 100 µm). (D, E) Quantitative analysis of positive area percent of PR (D) and ER (E) in injured endometrium among different treatment groups (n = 4 per group). (F) Quantitative analysis of vessel number in injured endometrium among different treatment groups (n = 3 per group). (G) Representative Western blot images and quantitative analysis of endometrial receptivity markers VEGFA (H) and ITGβ3 (I) in injured endometrium among different treatment groups ("−": control side; "+": damage side) (n = 4 per group). Results are presented as mean ± SEM. P < 0.05, **P < 0.01.

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  Adequate endometrial angiogenesis is essential for tissue reconstruction. Our study demonstrated that angiogenesis in the damaged side was significantly impaired compared to that in the control side. However, biotin-pep/IGFp hydrogel treatment enhanced angiogenesis, as indicated by an increase in the number of CD31/α-smooth muscle actin (α-SMA) + vessels (Figs. 3C and F, Fig. S11 in Supporting information). Additionally, the increased expression of Ki67 in the biotin-pep/IGFp hydrogel group suggested an enhanced capacity for tissue repair (Fig. S12 in Supporting information). Moreover, we assessed endometrial receptivity markers using western blotting. In the damaged endometrium (Figs. 3G–I), the levels of vascular endothelial growth factor A (VEGFA) and integrin β3 (ITG-β3) were significantly reduced than those in the control. Following hydrogel treatment, the expression levels of VEGFA and ITG-β3 significantly increased in the damaged tissue. Biotin-pep/IGFp hydrogel treatment effectively restored their expression, highlighting its potential to enhance endometrial receptivity.

  The primary function of the endometrium is to facilitate embryo implantation and support its development. Female rats were mated with fertile males 14 days post-surgery to assess the functional recovery of damaged endometrium. The embryo implantation rate in the injured endometrium was significantly lower compared to the control side. However, following biotin-pep/IGFp hydrogel treatment, the pregnancy rate increased demonstrating that the hydrogel injection effectively restored the functional capacity of the damaged endometrium (Fig. 4). The growth-promoting characteristics of the biotin-pep/IGFp hydrogel highlight its potential as a therapeutic option for treating IUA, enhancing both the structural and functional recovery of the endometrium.

  Figure 4

  

  Figure 4.  Biotin-pep/IGFp hydrogel improves fertility in the IUA rat model. Representative images of uteri with implantation sites on pregnancy day 15 (A) and quantitative analysis of pregnancy rate (B), and embryo numbers (C) in different treatment groups. Red arrows indicate the injury of the uterus. Black arrows indicate cervix. Results are presented as mean ± SEM (n = 4 per group). **P < 0.01.

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  Endometrial injury can significantly compromise endometrial function and fertility. This study proposes the use of a biotinylated peptide hydrogel loaded with IGFp as an innovative therapeutic approach. Due to its excellent mechanical properties and stability, the biotinylated self-assembled peptide hydrogel serves as an effective carrier of IGFp. Additionally, its biocompatibility and bioactivity ensure integration into biological systems, delivery of IGFp to affected endometrial tissues, and gradual release of IGFp for therapeutic effects. Experiments using the IUA rat model demonstrated that biotin-pep/IGFp hydrogel treatment enhanced glandular regeneration and vascular restoration, reduced collagen deposition, and facilitated restoration of the structural and functional integrity of the endometrium. Additionally, this hydrogel enhanced endometrial receptivity by facilitating the recovery of PRs, which are crucial for successful conception and pregnancy maintenance. These findings highlight the transformative potential of the two-component peptide hydrogel in treating IUA. It not only restores the endometrial structure but also enhances the biochemical environment required for conception, offering a promising solution for enhancing the fertility in patients with IUA.

  Declaration of competing interest

  The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

  CRediT authorship contribution statement

  Weiqi Zhang: Writing – original draft, Visualization, Validation, Methodology, Formal analysis, Data curation. Hang Wu: Methodology, Investigation, Formal analysis, Data curation. Limin Xie: Methodology, Investigation, Formal analysis, Data curation. Yixin Liang: Visualization, Investigation, Data curation. Xiaowan Huang: Investigation, Data curation. Zhimou Yang: Investigation, Funding acquisition, Data curation. Tengyan Xu: Writing – review & editing, Supervision, Resources, Project administration, Funding acquisition, Conceptualization. Feng Lin: Writing – review & editing, Supervision, Resources, Project administration, Funding acquisition, Conceptualization.

  Acknowledgments

  F. Lin acknowledges the financial support from the Zhejiang Provincial Natural Science Foundation of China (No. LBY24H040012), and Discipline Cluster of Oncology of Wenzhou Medical University (No. z1-2023007). T. Xu acknowledges the financial support from the National Natural Science Foundation of China (No. 32401107). Z. Yang acknowledges the financial support from the Discipline Cluster of Oncology of Wenzhou Medical University (No. z3-2023027).

  Supplementary materials

  Supplementary material associated with this article can be found, in the online version, at doi: 10.1016/j.cclet.2024.110800.

  
  
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  Figure 1  Design and characterization of the two-component peptide hydrogel (A) Chemical structure of Biotin-pep. (B, C) Optical images of Biotin-pep hydrogel before and after adding IGFp. (D, E) TEM morphology of Biotin-pep hydrogel before and after adding IGFp. (F) Dynamic frequency sweep of Biotin-pep hydrogel before and after adding IGFp. (G) Fourier transforms infrared spectroscopy Biotin-pep hydrogel before and after adding IGFp. (H) Detection of cycling breaking and recovery ability of Biotin-pep/IGFp hydrogel.

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  Figure 2  Biotin-pep/IGFp hydrogel promotes the regeneration of injured endometrium in vivo experiment. (A) Schematic diagram of animal study design. (B) Representative H & E-stained images of endometrium for each group. (C) Representative Masson-stained images of endometrium for each group (scale bar: upper image, 500 µm; lower image, 200 µm). (D–F) Relative gland number (D), relative thickness (E), and relative fibrosis rate (F) of injured endometrium compared to self-controls in each group. Results are presented as mean ± SEM (n = 4 per group). P < 0.05, **P < 0.01. n.s., no significance.

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  Figure 3  Biotin-pep/IGFp hydrogel improve endometrial receptivity in vivo experiment of injured endometrium. (A, B) Representative immunohistochemical staining of progesterone receptors (A) and estrogen receptors (B) in injured endometrium among different treatment groups. scale bar: upper image, 100 µm; lower image, 50 µm. (C) Relative quantity of blood vessels in damaged endometrium of different treatment groups (blue: 2-(4-amidinophenyl)−6-indolecarbamidine dihydrochloride (DAPI); green: CD31; red: α-SMA. Scale bar: 100 µm). (D, E) Quantitative analysis of positive area percent of PR (D) and ER (E) in injured endometrium among different treatment groups (n = 4 per group). (F) Quantitative analysis of vessel number in injured endometrium among different treatment groups (n = 3 per group). (G) Representative Western blot images and quantitative analysis of endometrial receptivity markers VEGFA (H) and ITGβ3 (I) in injured endometrium among different treatment groups ("−": control side; "+": damage side) (n = 4 per group). Results are presented as mean ± SEM. P < 0.05, **P < 0.01.

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  Figure 4  Biotin-pep/IGFp hydrogel improves fertility in the IUA rat model. Representative images of uteri with implantation sites on pregnancy day 15 (A) and quantitative analysis of pregnancy rate (B), and embryo numbers (C) in different treatment groups. Red arrows indicate the injury of the uterus. Black arrows indicate cervix. Results are presented as mean ± SEM (n = 4 per group). **P < 0.01.

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