Seamless vascularized large-diameter tubular collagen scaffolds reinforced with polymer knittings for esophageal regenerative medicine.

A clinical demand exists for alternatives to repair the esophagus in case of congenital defects, cancer, or trauma. A seamless biocompatible off-the-shelf large-diameter tubular scaffold, which is accessible for vascularization, could set the stage for regenerative medicine of the esophagus. The use of seamless scaffolds eliminates the error-prone tubularization step, which is necessary when emanating from flat scaffolds. In this study, we developed and characterized three different types of seamless tubular scaffolds, and evaluated in vivo tissue compatibility, including vascularization by omental wrapping. Scaffolds (luminal Ø âˆ¼ 1.5 cm) were constructed using freezing, lyophilizing, and cross-linking techniques and included (1) single-layered porous collagen scaffold, (2) dual-layered (porous+dense) collagen scaffold, and (3) hybrid scaffold (collagen+incorporated polycaprolacton knitting). The latter had an ultimate tensile strength comparable to a porcine esophagus. To induce rapid vascularization, scaffolds were implanted in the omentum of sheep using a wrapping technique. After 6 weeks of biocompatibility, vascularization, calcification, and hypoxia were evaluated using immunohistochemistry. Scaffolds were biocompatible, and cellular influx and ingrowth of blood vessels were observed throughout the whole scaffold. No calcification was observed, and slight hypoxic conditions were detected only in the direct vicinity of the polymer knitting. It is concluded that seamless large-diameter tubular collagen-based scaffolds can be constructed and vascularized in vivo. Such scaffolds provide novel tools for esophageal reconstruction.

Culture of ovine esophageal epithelial cells and in vitro esophagus tissue engineering.

BACKGROUND: Esophagus replacement presents major surgical challenges both in the pediatric and in adult patients since the various surgical techniques presently employed are associated with complications and high morbidity. AIM: The aim of this study was to establish protocols for isolation and culture of ovine esophageal epithelial cells (OEEC) and to investigate their viability on collagen scaffolds for in vitro tissue engineering. METHODS: OEEC were sourced from adult Austrian mountain sheep. Briefly, the esophagus was dissected, treated with dispase to separate the epithelial layer, and further subjected to a modified explants technique to isolate OEEC. The OEEC were cultured in vitro and seeded on to unidirectional two-dimensional and three-dimensional collagen scaffolds. RESULTS: Successful protocol was established for OEEC isolation and culture. OEEC exhibited organization and differentiation after 7 days in culture, which was complete after 18 days with the formation of a single layer sheet of differentiated cells exhibiting morphology of mature esophageal epithelium. OEEC seeded on two-dimensional collagen scaffolds demonstrated viability up to 6 weeks of in vitro culture with single layer epithelium formation after 3 weeks confirmed using pan-Cytokeratin markers. OEEC on three-dimensional scaffolds were viable for 6 weeks but did not form an epithelium sheet. CONCLUSION: Protocols for OEEC isolation were developed and established from adult ovine esophageal tissue. The generation of sheets of esophageal epithelium in culture and the viability of OEEC on collagen scaffolds for 6 weeks in vitro was observed. The prerequisite for esophagus tissue engineering, which is the ability to form epithelium when seeded on collagen scaffolds, was demonstrated.