A Decellularized Uterine Endometrial Scaffold Enhances Regeneration of the Endometrium in Rats

Abstract

Partial or whole regeneration of the uterine endometrium using extracellular matrix (ECM)-based scaffolds is a therapeutic strategy for uterine infertility due to functional and/or structural endometrial defects. Here, we examined whether the entire endometrium can be regenerated circumferentially using an acellular ECM scaffold (decellularized endometrial scaffold, DES) prepared from rat endometrium. We placed a silicone tube alone to prevent adhesions or a DES loaded with a silicone tube into a recipient uterus in which the endometrium had been surgically removed circumferentially. Histological and immunofluorescent analyses of the uteri one month after tube placement revealed more abundant regenerated endometrial stroma in the uterine horns treated with tube-loaded DES compared to those treated with a tube alone. Luminal and glandular epithelia, however, were not fully recapitulated. These results suggest that DES can enhance the regeneration of endometrial stroma but additional intervention(s) are needed to induce epithelization. Furthermore, the prevention of adhesions alone allowed the endometrial stroma to regenerate circumferentially even without a DES, but to a lesser degree than that with a DES. The use of a DES together with the prevention of adhesions may be beneficial for efficient endometrial regeneration in the uterus that is largely deficient of endometrium.

Keywords: ECM (extracellular matrix); endometrium; regenerative medicine; scaffold.

Figure 1

Isolation and characterization of the entire rat endometrium. (A) The entire endometrium (E) was separated and peeled off from the outer layer of the uterus containing myometrium (M) (left panel). A representative macroscopic image of the peeled-off E, separated M, and the whole uterine horn (E + M) is shown in the panel on the right. (B) As a positive control, the whole uterus was cross-sectioned and used for histology with hematoxylin and eosin (H&E) staining (left-most panel) and immunofluorescence staining using antibodies against cytokeratin (CK, green), vimentin (Vm, green), and smooth muscle actin (SMA, green) as indicated. (C) The peeled endometrium and (D) remaining outer layer of the uterus was cross-sectioned and analyzed as described in (B). Scale bars, 500 µm.

Figure 2

Preparation and characterization of decellularized rat endometrial scaffolds (DES). (A) The excised cylindrical endometrium was cut into 1.5 cm long pieces. (B–H) Endometrial pieces before (left panels) and after (right panels) decellularization were cross-sectioned and subjected to H&E staining (B) and immunofluorescence staining using antibodies against collagen type I (Col) (C), laminin (Lam) (D), Hoechst (Ho) (E), CK (F), Vm (G), and SMA (H). Scale bars, 500 µm.

Figure 3

Placement of a silicone tube with or without a DES into endometrium-defective uterus in rats. (A) A silicon tube (white arrow) inserted into the lumen of cylindrical DES. (B) A tube alone (T) or tube-loaded DES (T+D) was placed into the endometrium-defective site of each uterine horn (circled by white and yellow dotted lines, respectively). The muscle layer and serosa were continuously sutured.

Figure 4

In vivo regeneration of the endometrium, following the placement of a silicone tube alone or a tube-loaded DES (T+D) in rats. (A) Representative image of the uterus excised from the rat treated for one month, showing that each horn treated with T+D (white arrowhead) or T (yellow arrowhead) was grossly normal. (B–D) Uterine horn that underwent no treatment (Normal) or the indicated treatment was cross-sectioned and subjected to H&E staining (B) or immunofluorescence staining using antibodies against CK (C), SMA ((D), upper panels), and Vm ((D), middle panels). Nuclei were stained with Hoechst (Ho). Scale bars, 500 µm.

Figure 5

Quantitative analysis of regeneration potentials of silicone tube only (T) and tube-loaded DES (T+D). Based on the assumption that regeneration potential positively correlates with the area and cell (nuclei) number of the newly generated stroma, the total uterus/stroma ratio for these two parameters was calculated (A,B), as described in the text. The mean values of the relative area and nuclear number ratios obtained from the untreated normal tissue were 0.513 and 0.444, respectively. Values are expressed as mean ± SEM together and shown in dot plots of individual values obtained from the five independent experiments. * p < 0.05.

Figure 6

Overview of experimental design and procedures. The uterus of a donor rat was excised, and the endometrium (E) was separated from the outer layer, which consists mainly of myometrium (M), of the uterus. The separated E was decellularized and designated as a decellularized endometrium scaffold (DES). The recipient rat underwent the same separation of E but without excision of the uterus, such that a 1.5 cm long section of E alone was removed and the M of both uterine horns remained. A silicone tube or a tube-loaded DES was placed into the endometrium-deficient section of each uterine horn, and the outer muscle layer was then closed with continuous suturing. The silicone tube was used to prevent intrauterine adhesion. A month later the uterus was excised for histological and immunofluorescence microscopic analyses. The number of rats used as indicated are shown in parentheses.