L. Y. Martin1, M. Ladd1, H. Jia1, C. Sodhi1, A. Werts1, Y. Yamaguchi1, D. Nino1, P. Lu1, Q. Zhou1, J. Sung1, W. Fulton1, T. Prindle1, E. Banfield1, D. Hackam1 1Johns Hopkins University School Of Medicine,Surgery,Baltimore, MD, USA
Introduction:
Short bowel syndrome is a leading cause of morbidity and mortality in children for which treatment options are limited. Several investigators have reported successful techniques for growing intestinal stem cells (“enteroids”) in vitro, and we recently reported successful mucosal growth on a novel scaffold in dogs. We now propose to test the feasibility and functionality of implantation of scaffold with auto-transplanted intestinal stem cells in a porcine model that resembles the human disease in infants.
Methods:
Synthetic scaffolds with a 3D architecture that resembles the human intestine were prepared with a serial fabrication technique, using laser indentation in an agarose gel to create microvilli. On day of life 3, piglets (1500±50g, n=5) underwent laparotomy and small bowel resection (10 cm.) Intestinal stem cells (ISCs) were isolated by mucosal stripping and cellular disaggregation from the resected specimen, and cultured on collagen-coated scaffold with serum at 37oC. Immunohistochemistry was used to confirm growth and differentiation of intestinal tissue on the scaffold. At 8 days of age, 4 piglets underwent laparotomy with auto-transplantation of enteroid-seeded scaffold into the omentum. Four control conditions were utilized to account for variables: no operation (n=1), small bowel resection alone (n=2), small bowel resection and empty scaffold implantation in single operation (n=1), small bowel resection with delayed implantation of empty scaffold on DOL 8 (n=1). Repeat laparotomy was performed at age 26d (n=2) and 31 (n=1), and the scaffold was evaluated grossly for vascularization. H&E and IHC staining were used to evaluate cellular ingrowth and vascularization.
Results:
The operative model was well-tolerated. Weight gain was appropriate with 100-150% increase by 2 weeks. Immunohistochemistry revealed proliferation of the stem cells on the scaffold villi with staining for Ki67, BRDU, and PCNA, and differentiation via staining for Muc2a, SI, Lysozyme, and chromogranin. After implantion into the piglet omentum, subsequent laparotomy revealed that the scaffold was encapsulated by 15 days, with gross evidence of vascularization as revealed by fluorescein angiography. Confocal microscopic evaluation confirmed the expression of vascular markers PECAM and Endothelin on the scaffold, as well as smooth muscle markers actin and vimentin
Conclusion:
We have demonstrated successful growth intestinal tissue on a scaffold, and establishment of a blood supply in a large animal model resembling the human condition. These findings raise the possibility of tissue engineering for intestinal stem cells in the management of children with short bowel syndrome
