In vitro evaluation of bi-layer silk fibroin scaffolds for gastrointestinal tissue engineering

Silk fibroin scaffolds were investigated for their ability to support attachment, proliferation, and differentiation of human gastrointestinal epithelial and smooth muscle cell lines in order to ascertain their potential for tissue engineering. A bi-layer silk fibroin matrix composed of a porous silk fibroin foam annealed to a homogeneous silk fibroin film was evaluated in parallel with small intestinal submucosa scaffolds. AlamarBlue analysis revealed that silk fibroin scaffolds supported significantly higher levels of small intestinal smooth muscle cell, colon smooth muscle cell, and esophageal smooth muscle cell attachment in comparison to small intestinal submucosa. Following 7 days of culture, relative numbers of each smooth muscle cell population maintained on both scaffold groups were significantly elevated over respective 1-day levels-indicative of cell proliferation. Real-time reverse transcription polymerase chain reaction and immunohistochemical analyses demonstrated that both silk fibroin and small intestinal submucosa scaffolds were permissive for contractile differentiation of small intestinal smooth muscle cell, colon smooth muscle cell, esophageal smooth muscle cell as determined by significant upregulation of α-smooth muscle actin and SM22α messenger RNA and protein expression levels following transforming growth factor-β1 stimulation. AlamarBlue analysis demonstrated that both matrix groups supported similar degrees of attachment and proliferation of gastrointestinal epithelial cell lines including colonic T84 cells and esophageal epithelial cells. Following 14 days of culture on both matrices, spontaneous differentiation of T84 cells toward an enterocyte lineage was confirmed by expression of brush border enzymes, lactase, and maltase, as determined by real-time reverse transcription polymerase chain reaction and immunohistochemical analyses. In contrast to small intestinal submucosa scaffolds, silk fibroin scaffolds supported spontaneous differentiation of esophageal epithelial cells toward a suprabasal cell lineage as indicated by significant upregulation of cytokeratin 4 and cytokeratin 13 messenger RNA transcript levels. In addition, esophageal epithelial cells maintained on silk fibroin scaffolds also produced significantly higher involucrin messenger RNA transcript levels in comparison to small intestinal submucosa counterparts, indicating an increased propensity for superficial, squamous cell specification. Collectively, these data provide evidence for the potential of silk fibroin scaffolds for gastrointestinal tissue engineering applications.