Tissue-Specific Effects of Esophageal Extracellular Matrix

Abstract

Biologic scaffolds composed of extracellular matrix (ECM) have been used to facilitate repair or remodeling of numerous tissues, including the esophagus. The theoretically ideal scaffold for tissue repair is the ECM derived from the particular tissue to be treated, that is, site-specific or homologous ECM. The preference or potential advantage for the use of site-specific ECM remains unknown in the esophageal location. The objective of the present study was to characterize the in vitro cellular response and in vivo host response to a homologous esophageal ECM (eECM) versus nonhomologous ECMs derived from small intestinal submucosa and urinary bladder. The in vitro response of esophageal stem cells was characterized by migration, proliferation, and three-dimensional (3D) organoid formation assays. The in vivo remodeling response was evaluated in a rat model of esophageal mucosal resection. Results of the study showed that the eECM retains favorable tissue-specific characteristics that enhance the migration of esophageal stem cells and supports the formation of 3D organoids to a greater extent than heterologous ECMs. Implantation of eECM facilitates the remodeling of esophageal mucosa following mucosal resection, but no distinct advantage versus heterologous ECM could be identified.

FIG. 1.

Preparation and characteristics of extracellular matrix (ECM) scaffolds. (A) Overview of decellularization process for preparing urinary bladder matrix (UBM), small intestinal submucosa (SIS), and eECM. (B) Gel chromatography of ECM materials showing features in banding patterns of different ECM materials. Color images available online at www.liebertpub.com/tea

FIG. 2.

Migration of esophageal stem cells. (A) Representative images of 4′,6-diamidino-2-phenylindole (DAPI)-stained migrating cells toward varying concentrations of ECM. (B) Quantification of migrated cells in response to ECM scaffolds. **p<0.01.

FIG. 3.

Capacity of ECM hydrogels to support organoid formation. (A) Comparison number of organoids formed in different ECM types. Data are normalized to the number of organoids formed in SIS. (B) Comparison of number of organoids formed in ECM at 2 and 6 mg/mL. Data are normalized to the number of organoids present at 2 mg/mL concentration of ECM. **p<0.01.

FIG. 4.

Cytokeratin immunolabeling. (A) Representative images of organoids formed in eECM. (B) Representative image of normal esophageal mucosa. Cytokeratin 14, a basal epithelial cell marker, is stained red. Cytokeratin 13, a marker of suprabasal epithelial cells, is stained green. Nuclei (DAPI) is shown blue. Scale bars = 50 μm. Color images available online at www.liebertpub.com/tea

FIG. 5.

Proliferation of organoid cells. (A) Representative images of EdU-stained organoids following 2-h EdU exposure. EdU⁺ cells are shown in red. Nuclei are shown in blue. (B) Quantification of number of cells per organoid and number of EdU⁺ cells per organoid. Scale bar = 50 μm. Color images available online at www.liebertpub.com/tea

FIG. 6.

Histology and immunolabeling of explants at 14 days postsurgery. The in vivo host response to no treatment (A, D), UBM scaffold (B, E), and eECM (C, F) was assessed histologically with hematoxylin and eosin (H&E) staining and by immunolabeling for stratified squamous epithelium (cytokeratin 14, green). Blood vessel endothelial cells stain positive for cytokeratin 14. Arrows indicate positive staining and scale bars = 100 μm. Color images available online at www.liebertpub.com/tea