Esophageal epithelium regeneration on fibronectin grafted poly(L-lactide-co-caprolactone) (PLLC) nanofiber scaffold.

In order to mimic normal epithelium regeneration on synthetic scaffold in vitro, biodegradable elastic poly(l-lactide-co-caprolactone) (PLLC) was processed into nanofibrous scaffold using electrospinning technology. An adhesive protein, fibronectin (Fn), was grafted onto the scaffold fiber surface via a two-step reaction: polyester aminolysis followed by Fn coupling via glutaraldehyde. Tensile testing was performed to measure the effect of aminolysis on the scaffold mechanical properties. The strain decreased but the tensile strength remained almost constant after aminolysis. However, no obvious difference of the nanofiber surface morphology was found after Fn grafting using scanning electron microscopy (SEM). Porcine esophageal epithelial cells were seeded on the Fn bonded scaffold to test the cell growth promotion against the control unmodified PLLC nanofiber scaffold using tissue culture polystyrene (TCPS) plate as a reference. Anti-cytokeratin AE1/AE3 was used as the primary antibody to confirm the esophageal epithelial phenotype. SEM observation, immunostaining and Western Blotting to compare the collagen type IV synthesis showed that the Fn grafted on PLLC scaffold greatly promotes epithelium regeneration. This modified scaffold is expected to be a good candidate for functional esophagus substitutes.

Dual requirements of extracellular matrix protein and chitosan for inducing adhesion contact evolution of esophageal epithelia.

It has been recently shown that chitosan (CHI)/collagen prostheses induced epithelization at the esophagus site of animal model. However, little is known on the biophysical mechanisms of cell adhesion on CHI-based material pertaining to esophagus tissue engineering. In this study, the adhesion contact dynamics of porcine esophageal epithelial cells seeded on CHI surface is probed using confocal-reflectance interference contrast microscopy in conjunction with phase-contrast microscopy. First of all, cells fail to form any adhesion contact on either CHI or elastin (ES)-coated surface. On CHI coated with fibronectin (CHI-FN) or elastin (CHI-ES), strong adhesion contact of cells evolved over time until they reached a steady-state level. The initial cell deformation rates of cells on CHI-FN and CHI-ES are 0.0138 and 0.0151 min(-1), respectively. Interestingly, cells on fibronectin (FN) coated substrate transiently form strong adhesion contact and eventually undergo deadhesion. Moreover, the steady-state adhesion energy of epithelial cells on CHI-FN is 1.73 and 148 times larger than that on CHI-ES and FN, respectively. The actin of cells on CHI-FN transforms from microfilament meshes at cell periphery to stress fibers throughout the cytoplasm during cell seeding. At the same time, vinculin staining demonstrated the evolution of focal adhesion complexes in cells on CHI-FN after 130 min of seeding. Interestingly, CHI-ES induces the formation of focal adhesion complexes in a lesser extent in cell but fails to lead to stress fiber formation. Overall, our study reveals that long-term adhesion contact evolution of esophageal epithelia is only triggered by both extracellular matrix protein and chitosan.

Effect of electrospun poly(D,L-lactide) fibrous scaffold with nanoporous surface on attachment of porcine esophageal epithelial cells and protein adsorption.

Electrospun scaffolds have been increasingly used in tissue engineering applications due to their size-scale similarities with native extracellular matrices. Their inherent fibrous features may be important in promoting cell attachment and proliferation on the scaffolds. In this study, we explore the technique of fabricating electrospun fibers with nano-sized porous surfaces and investigate their effects on the attachment of porcine esophageal epithelial cells (PEECs). Porosity was introduced in electrospun poly(D,L-lactide) fibers by creating vapor-induced phase separation conditions during electrospinning. The nanoporous fiber scaffolds were mechanically weaker than the conventional solid fiber scaffolds and solvent-cast films of the same polymer. However, the nanoporosity of the fibers was found to enhance the levels of adsorbed protein from a dilute solution of fetal bovine serum. The amount of protein adsorbed by nanoporous fiber scaffolds was approximately 80% higher than the solid fiber scaffolds. This corresponds to an estimated 62% increase in surface area of the porous fibers than the solid fibers. By comparison, the solvent-cast films adsorbed low levels of protein from the FBS solution. In addition, the porous fibers were found to be advantageous in enhancing initial cell attachment as compared with the solid fibers and solvent-cast films. It was observed that nanoporous fiber scaffolds seeded with PEECs had significantly greater number of viable cells attached than the solid fiber scaffolds after 10 and 24 h in culture. Hence, our results indicate that nanosized porous surfaces on electrospun fibers enhance both protein adsorption and cell attachment. These findings provide a method to improve cell-matrix interactions of electrospun scaffolds for tissue engineering applications.