Hybrid endometrial-derived hydrogel and human endometrial organoids synergize for uterine regeneration in an immunocompetent murine model

Abstract

Background The human endometrium is a regenerative tissue essential for fertility, but pathological conditions like Asherman syndrome, endometrial atrophy, and thin endometrium can impair its function. Current therapies lack efficacy, driving demand for innovative regenerative therapies. In this context, endometrial-derived hydrogels and organoids have shown promise individually for tissue regeneration, but their combined therapeutic potential has not been previously evaluated in vivo. This study explores a dual regenerative strategy combining a hybrid hydrogel — composed of synthetic PuraMatrix® and endometrial extracellular matrix hydrogel — with human endometrial organoids in an immunocompetent murine model with uterine damage. Methods Endometrial damage model was established in female C57BL/6 mice (n = 46) via uterine injury using 70° ethanol. After 4 days of endometrial damage, human endometrial organoids were co-injected with the hybrid hydrogel into the uterine horns. Two weeks post-injection, a subset of mice (n = 25) was sacrificed for biocompatibility, histological, and transcriptomic analyses. Functional recovery of the endometrium was assessed in the remaining animals (n = 21) through fertility outcome evaluation. For endometrial regeneration analyses, normally distributed data were analyzed by one-way ANOVA and Tukey’s multiple comparisons, while non-normally distributed data were analyzed by the Kruskal–Wallis test with Dunn’s multiple comparisons. For fertility outcomes, t-test or Mann–Whitney U tests for 2-by-2 comparisons were performed.

1. Introduction

The human endometrium is the innermost layer of the uterus, undergoing tissue regeneration every menstrual cycle [1]. This dynamic tissue consists of luminal and glandular epithelia surrounded by stroma and blood vessels. Additionally, the endometrium is supported by a niche of epithelial and stromal stem cells that contribute to its regenerative capacity [2]. This remarkable ability to renew itself is essential for reproductive success, but pathological changes in the endometrial structure and function can lead to infertility. Endometrial diseases, such as Asherman syndrome (AS), endometrial atrophy (EA), and thin endometrium (TE) are characterized by fibrosis, glandular loss, and impaired vascularization, which significantly reduce the chances of a successful pregnancy [3]. The limited effectiveness of current treatments, including hormonal therapies and surgical interventions, highlights the need for innovative therapies to restore the endometrial structure and function [4].

In recent years, regenerative medicine has emerged as a promising strategy for endometrial repair [4]. Within this field, bioengineering approaches, particularly the use of hydrogels, have been explored for their potential to support tissue regeneration [5]. Natural hydrogels, such as those derived from the endometrial extracellular matrix (EndoECM), can be obtained by the decellularization of the endometrium to remove cellular components while preserving the native proteins and bioactive factors necessary for tissue remodelling [6]. On the other hand, synthetic hydrogels, such as PuraMatrix® (PM, Corning), offer excellent biocompatibility, high mechanical stability, and durability [5, 7]. Due to its simple composition, consisting exclusively of water and RADA16 peptide sequences, several PM-based formulations have been approved in Europe, the United States, and Japan to control intraoperative bleeding in surgical procedures [8]. RADA16 is a peptide-based biomaterial whose acidic aqueous solution spontaneously forms an extracellular matrix-like three-dimensional structure [9]. While ECM-derived hydrogels retain the biochemical cues of the native endometrium, they often lack mechanical stability, whereas synthetic hydrogels provide structural support but lack biological signals. Therefore, combining these two types of hydrogels into a hybrid system could synergistically enhance endometrial regeneration.