**5. Clinical potential and future perspectives**

(**Figure 4**). Silk fibroin is known to have good physical and mechanical properties, and also good biocompatibility. Electrospun fibroin scaffolds together with optimal pore parameters and protein coating extracted from animal esophagus could be great candidates in esopha‐

The muscle tissue of esophagus consists of striated muscle (skeletal) in the upper third, mix‐ ture of skeletal and smooth muscle in the middle third, and pure smooth muscle in the lower third. These muscle contents arrange into endo‐circular and exo‐longitudinal sub‐bilayers to play an important role in propelling the swallowed food or fluid into the stomach via mus‐ cle peristalsis. Generating an oriented muscle architecture to mimic the tissue of muscularis externa is an important issue to restore the functions of tissue‐engineered esophagus. Many researchers studied the relationship between scaffold's chemistry and microstructure and muscle cells' phenotype. Stegemann once verified that the behaviors of smooth muscle cell (SMC) were positively correlated to the scaffold geometry (2D and 3D) [60]. Li et al. believed that the scaffold geometry played an important role in modulating SMC phenotype. They cultured SMCs and discovered that cells in 3D collagen (type I) gels had lower proliferation and higher collagen synthesis than the cells in 2D collagen substrate [61]. Chan‐Park verified that smooth muscle α‐actin of SMCs cultured in microchannels upregulated greatly, sug‐ gesting a phenotype shift from synthetic to contractile state of cells [62]. They thus believed that 3D microchannels could encourage cells to reorganize into orientation patterns because SMC have a natural self‐arrangement propensity. Moreover, the narrow space of channels around 100 μm or less helped cells to achieve more uniform orientation. We also fabricated scaffolds with circular and longitudinal microchannel patterns (**Figure 5**). Further, the scaf‐ fold surface was grafted with silk fibroin using our method of diamine aminolysis and GA crosslinking. The primary esophageal SMC was cultured in these 3D protein‐grafted chan‐ nels in order to achieve SMC phenotype regulation and *in situ* muscle formation [63]. The results confirmed that primary esophageal smooth muscle cells exhibited fine alignment in all types of microchannels while SMCs in the interval channels communicated well through

Some researchers had considered and investigated that mechanical stimulation might be an effective way to regulate SMC phenotype. Ritchie et al. designed a system to exert mechanical forces on esophageal smooth muscle cells. They discovered that cells on the flexile polyure‐ thane membrane displayed alignment parallel to the force direction when low cyclic strains (2%) was used, but alignment perpendicular to the force direction when high strains (5 and 10%) used [64]. Cha et al. reported that muscle cells would orient according to the optimal movement of the tissue. They adopted cyclic mechanical strain (a homemade stretching chamber) on primary myofibroblasts, and promoted the cell differentiation, and further mod‐ ulated the orientation and proliferation of the differentiated smooth muscle cell. Their conclu‐ sion was that myofibroblast/scaffold hybrids with cyclic strain could be applied to organize

**4. Constitution of muscularis propria of the esophagus**

geal tissue engineering [59].

184 Esophageal Abnormalities

the gaps (**Figure 5**).

smooth muscle cells with muscle tissue functions [65].

With the development of stem cell technology, some kinds of stem cells, for example, embry‐ onic stem cell, mesenchymal stem cell, progenitor stem cell, induced pluripotent stem cell, etc., are adopted to be the seeded cells in tissue reconstruct. In case of esophagus, bone mes‐ enchymal stem cell (bMSC) is more often used to seed on scaffolds than other kinds of stem cells to regenerate or remodel the engineered esophagus. Taylor and Macchiarin reported that allogeneic mesenchymal stromal cells were seeded on the decellularized rat esophagi to orthotopically replace the entire cervical esophagus. After 14 days, the explanted grafts showed regeneration of all the major cell and tissue components of the esophagus including functional epithelium, muscle fibers, nerves, and vasculature. Thus, this tissue‐engineered esophageal scaffold was considered as a significant step toward the clinical application of bioengineered esophagi [66].

In summary, the research of substrate materials and scaffold fabrications in esophageal tissue engineering has made great progress in past decades. Esophagus repairs in animal models and even clinical tests are being attempted and the techniques are being improved. Materials with appropriate physical and chemical properties are still being developed. Optimizing scaffolds and cells for epithelium regeneration or/and muscle constitution, and their combination have been in progress. Some crucial problems, such as complications from stricture to dilation, angiogenesis and innervation consideration, little or no muscle regeneration in the implants, etc., need to be issued before the tissue‐engineered esophagus can be a viable conduit for surgical replacement in clinic. And, graft‐to‐host integration and remodeling of the organ functions like peristalsis and nerve guide would be the important gauge for the success in tissue engineering of esophagus. With the development of mate‐ rial science and engineering, stem cell biology, and other related theories and technologies, tissue‐engineered esophagus is able to foresee the promising employment in clinic in near future.
