Constitution And Cytocompatibility Of Artificial Epithelium For Esophageal Tissue Engineering Research

Esophageal carcinoma becomes one of the most common causes of cancer-related deathsworldwide. More than90%of the esophageal cancers originate from the lining epithelium of theesophagus. There are two major subtypes of carcinomas, including squamous carcinoma, whicharises from the squamous epithelium, and adenocarcinoma which arises from the glandularepithelium of the esophagus. Adenocarcinoma also arises within the dysplastic columnarepithelium of the distal esophagus. Tissue engineering scaffolds is deemed to be a potential way forreplacement of lost or malignant esophageal epithelia in future. Fabrication of bionic extracellularmatrix scaffold using biomaterials with proper structure is one of key issues in tissue engineering.In this work, polycaprolactone (PCL) was processed into nanofibrous scaffolds usingelectrospinning technology while biodegradable elastic poly(L-lactide-co-caprolactone)(PLLC)was processed into asymmetrical3-D porous scaffold using thermal-induced phase separationmethod.PCL fibrous menbrane was prepared via electrospinning. In order to reduce the fiber diameter(to mimic the constitution of esophageal basement membrane) and improve the cytocompatibilityof PCL scaffold, silk fibroin (SF) was sneaked, and PCL/SF composite fibers were prepared byelectrospinnig process. The morphology of PCL, PCL/SF, SF were observed under scanningelectron microscope (SEM). The results showed that the average diameter of the fibers graduallyreduced from214.1nm to140.8nm with the introduction of SF content, which is almost the sameas the fibers from normal esophageal basement membrane. Comparing with the mechanicalproperties of PCL fibrous membranes, the PCL/SF composite fibers displayed the better elasticityand strength. For the degradation test during30d span, they exhibited slightly weight lost at thefirst15d. The results of the scaffold’s cytocompatibility to porcine esophageal epithelial cells(PEECs) showed that cells adhered and spread well on the PCL/SF scaffold with high proliferationrate. The immunofluorescence and western-blotting measurements presented that PCL/SFcomposite fibrous scaffold was more suitable for EMECs adhesion, proliferation anddifferentiation than those of PCL scaffold.In our previous study, the topological characteristic of porcine esophageal basementmembrane was analyzed and the major proteins were extracted and quantified. Herein, the protein extraction was used to modify the electrospun PCL membrane. In vitro culture of PEECs provedthat proteins-modified PCL surface (denoted coated-PCL) can more effectively support the cellgrowth, which will be promising for biocompatible scaffold to support epithelial cell growth andenhance epithelium regeneration.On the other hand, an asymmetrical porous tube made from PLLC copolymer was fabricatedusing thermally induced phase separation (TIPS) method towards regeneration of tissue engineeredesophageal epithelium. The scaffold in which there are pores with3-9μm diameter on the lumenand pores with6-18μm diameter in the bulk was to mimic the mucosa constitute of normalesophagus. The porcine primary esophageal epithelial cells were seeded on the tube lumen after itwas coated by fibronectin(Fn). Cells were evaluated using hematoxylin and eosin (H&E) stainingas well as immunofluorescence with anti-cytokeratin14to confirm the squamous esophagealepithelial phenotype. A confluent layer of epithelial cells was observed throughout the wholelumen face with multilayer of cells at some locations. The results show that this tubular scaffoldgreatly promotes epithelium regeneration.In summary, all results indicated that the scaffolds constituted by electrospinning and TIPSmethod imitated the structure of esophageal membrane. It provided a good external enviroment forcell adhesion and proliferation after protein coated. Therefore, these scaffolds hold tremendouspotential as artificial epithelium for esophageal tissue engineering and will be provided for animalexperiment, which is one of key works of esophageal tissue engineering research of our group.