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Production of A Three-Dimensional Polymeric Scaffold by Molding In A Natural Acellular Matrix From Rat Small Intestine

Sunday, October 21, 2012: 10:48 AM
Versailles Ballroom (Hilton Riverside)
Giorgia Totonelli1, Giovanni G. Giobbe2, Panagiotis Maghsoudlou1, Agostino Pierro1, Simon Eaton1, Nicola Elvassore2 and Paolo De Coppi1, (1)Surgery Unit, UCL Institute of Child Health and Great Ormond Street Hospital for Children, London, United Kingdom, (2)Department of Industrial Engineering, University of Padua, Padua, Italy

Purpose , Tissue engineering has the potential to represent a valid alternative to the repair or regeneration of normal tissue function in damaged organs. Decellularised intestine reproduces the morphometric characteristics of the gut but it is derived from cadavers. Development of 3D scaffolds that mimic the role of the extracellular matrix and guide cells to restore functional tissue may replace decellularised organs. The aim of this study was to fabricate a biocompatible polymer molding of decellularised rat small intestine and to study the cell viability on the obtained 3D polymeric scaffold.

Methods , After ethical committee approval, rat small intestines were decellularised with a detergent-enzymatic treatment, via both intestinal lumen and vascular tree, as previously described. The acellular matrix was copied by replica molding technique with Polydimethylsiloxane (PDMS, weight ratio elastomer/curing agent 10:1). Dehydrated intestines cut lengthwise and opened on a petri dish were treated overnight with hexamethyldisilazane vapor. Liquid degassed pre-polymer was casted on treated intestine and allowed to settle on the surface for 1 hour. After baking at 70C for 2 hours, acellular matrices were removed from the silicon layer to obtain a negative replica of the intestinal surface. Following treatment of the negative mold with (Tridecafluoro-1,1,2,2-Tetrahydrooctyl)-1-Trichlorosilane, degassed PDMS were casted on the negative replica and positive silicon scaffolds were obtained and studied with environmental scanning electron microscopy (ESEM). After UV-sterilization and coating with Matrigel, positive molds were seeded with differentiated Human Embryonic Stem Cells (hESC). After 14 days of culture, cells were fixed and stained for nuclei (Hoechst) and actin (phalloidin conjugated with 488nm fluorophore), and observed at fluorescence microscope (Leica DMI6000 B).

Results , A high-fidelity replica of the intestinal luminal surface was obtained by molding with polymer of the acellular matrix. ESEM of the polymer showed a good reproduction of the intestinal lumen surface with preservation of the morphology and the three-dimensionality of the natural scaffold (Fig). In particular, analysis of the polymer revealed the presence of leaf-shaped villi replica (blu arrow) and of crypts at their basis (red arrow). To test the cell viability, the polymer was seeded with hESCs-derived epithelial cells. Cells remained viable after 2 weeks and they showed a good adhesion and uniform distribution on the 3D surface of the polymer, in particular by the crypts area.

Conclusion , The present study successfully demonstrates the possibility of generating a 3D polymeric intestinal scaffold, which entirely reflects the morphology of the native acellular matrix and promotes cell attachment. Furthermore, this scaffold is reproducible and non-immunogenic. This novel approach for the fabrication of 3D polymeric scaffolds holds promise for tissue engineering applications.