**3. Fabrication of 3D scaffold for esophageal tissue engineering**

Biodegradable 3D scaffolds serve as analogues of extracellular matrix (ECM) in the engineered tissue or organ. Therefore, the scaffold's chemistry and macro and/or microscale architecture must be helpful to maintain cell's functions including cell‐matrix adhesion, cell‐cell adhe‐ sion, cell migration, proliferation, differentiation, etc. On the other hand, 3D scaffold should provide spatial cues for cell infiltration, so that cells are capable of integrating with the under‐ lining substrate. People are always seeking techniques to fabricate spatial scaffolds. Some technologies like foaming, porogen leaching, electrospinning, or other fiber processing, phase separation, 3D microprinting, etc. were developed to construct 3D porous scaffolds. In par‐ ticular, electrospinning technology and thermally induced phase separation (TIPS) method have been extensively studied to constitute 3D scaffolds in tissue engineering of esophagus.

#### **3.1. Phase separation**

Phase separation of polymeric materials is often induced by thermal alteration. That is called thermally induced phase separation, shortened as TIPS. TIPS is one of the most practical

**Figure 2.** (a) A tubular mold; (b) overview of the tubular scaffold made from crosslinking of PLEGDMA, PEG diacrylate and NIPAAm.

techniques to prepare 3D scaffolds with optimal pore profiles by modulation of process parameters. It was firstly proposed by Castro as early as in 1981 [45] and extended to scaf‐ fold making after commencing of tissue engineering [36, 46–48]. The TIPS procedure usually involves four steps: (1) polymer dissolution in some solvent, (2) occurrence of phase separa‐ tion, (3) polymer gelation, and (4) solvent extraction [49]. There are many advantages of TIPS over other traditional methods such as porogen leaching and foaming. For example, TIPS has the abilities to create a variety of pore structures by employing different parameters, and abroad applications for substrate sources because of its universality for various materials like crystal or noncrystal synthesized polymers [50, 51].

Zhu et al. prepared porous scaffolds with ≥100 μm pore size and good pore interconnectivity using TIPS technique [52, 53]. In order to simulate the ECM architecture, the original TIPS was modified, and thus a scaffold with an asymmetrical pore structure in a hierarchical order was created (**Figure 3**). The pore size on the scaffold surface was small, 1–10 μm, while that of the scaffold bulk was large, ≥100 μm (**Figure 3b**). Primary porcine epithelial cell was cultured on this asymmetrical scaffold lumen surface and primary fibroblast was cultured in the scaffold bulk. The coculture results verified that the bulk with large pores allowed fibroblast migration and infiltration while the lumen superficies with micropores supported the growth of epithelial cells and served as a barrier against fibroblast penetration. The immuno‐fluorescent staining (nuclei displayed as blue and keratin as green) of epithelial cells exhibited that several layers of epithelial cells had formed after *in vitro* culture for 14 days on the scaffold lumen (**Figure 3**, **a2** and **b2**). The *in vivo* test showed that a complete layer of epithelium was regenerated on porcine esophagus lumen while the scaffold was being degraded after implantation for 5 months [53].

Beckstead et al. prepared porous sheets with salt‐leaching/gas foaming method. The ammonium bicarbonate salt with size from 38–75 to 150–250 μm was used as the porogen reagent. A mix‐ ture of 10% chloroform and 90% ethanol was adopted as the polymer solvent. After dissolved in the solvent completely, the polymer solution was evaporated while 50% aqueous citric acid solution was used to initiate gas foaming accompanying with salt leaching [54]. They evaluated the cell (rat esophageal epithelial cell) behaviors on the scaffolds derived from natural material (exemplified AlloDerm), and synthetic materials like poly(lactic‐co‐glycolic acid) (PLGA) and PCL/PLLA. The results exhibited that AlloDerm scaffold had superior epithelial organization and stratification over other artificial scaffolds. Further modification to the artificial scaffolds would be a necessary way to polish their chemistry and to improve the cell behaviors.

#### **3.2. Electrospinning**

hydroxyl‐terminated prepolymers and further linking with hexamethylene diisocyanate. The final product, PEU with high Mw, was achieved under the catalyst of fluorinated iron acetate [43]. Zhu group investigated the polymerizing of ester‐containing monomers like lactide, cap‐

was the most efficient catalyst among them to yield products with high mono‐

), or iron (II) acetylacetonate Fe(acac)2

mer conversion and number average molecular weight [44]. Based on these studies, an oligo‐ mer, poly(ethylene glycol‐co‐lactide) dimethacrylate (PLEGDMA) was further synthesized via ring‐opening polymerization of L‐LA and polyethylene glycol (PEG) under Fe(acac)3

tiation. After cross‐linking with PEG diacrylate and NIPAAm, or with linear prepolyurethane in the homemade mold, biodegradable tubular scaffolds with good mechanical properties were fabricated (**Figure 2**). These scaffolds were verified to be good enough to support the

Biodegradable 3D scaffolds serve as analogues of extracellular matrix (ECM) in the engineered tissue or organ. Therefore, the scaffold's chemistry and macro and/or microscale architecture must be helpful to maintain cell's functions including cell‐matrix adhesion, cell‐cell adhe‐ sion, cell migration, proliferation, differentiation, etc. On the other hand, 3D scaffold should provide spatial cues for cell infiltration, so that cells are capable of integrating with the under‐ lining substrate. People are always seeking techniques to fabricate spatial scaffolds. Some technologies like foaming, porogen leaching, electrospinning, or other fiber processing, phase separation, 3D microprinting, etc. were developed to construct 3D porous scaffolds. In par‐ ticular, electrospinning technology and thermally induced phase separation (TIPS) method have been extensively studied to constitute 3D scaffolds in tissue engineering of esophagus.

Phase separation of polymeric materials is often induced by thermal alteration. That is called thermally induced phase separation, shortened as TIPS. TIPS is one of the most practical

**Figure 2.** (a) A tubular mold; (b) overview of the tubular scaffold made from crosslinking of PLEGDMA, PEG diacrylate

growth of porcine esophageal cells like epithelial, fibroblast, and muscle cell.

**3. Fabrication of 3D scaffold for esophageal tissue engineering**

), ethanol iron (Fe(OC2

H5 ) 3

, as the catalyst. The result was

), iron (III)

ini‐

rolactone, glycolic acid, etc. using ferric chloride (FeCl3

acetylacetonate (Fe(acac)3

**3.1. Phase separation**

and NIPAAm.

that Fe(acac)3

180 Esophageal Abnormalities

The technique of electrospinning was first proposed by Formhals in 1934 [55]. After that, it was gradually applied in diverse regions, for example, filtration industry, wound dressings, con‐ trolled drug releasing, and scaffold making in tissue engineering, and so on. In particular, this technique has gained popularity in tissue engineering fields, as a means of making scaffold. The fiber sheets obtained from electrospinning process possesses many features similar to natural ECM, for example, fibers were loosely connected with nano to microscale diameters; the sheet has high porosity and high surface area to volume ratio. Therefore, this technology became an interesting and valuable way to constitute scaffolds for esophageal tissue engineering.

**Figure 3.** Overview of tubular scaffold (a1) and cross‐section structure (b1). The scaffold was prepared with TIPS technique using biodegradable poly(l‐lactide‐co‐caprolactone) as the substrate material. Primary epithelial cells were cultured on the scaffold lumen for 14 days and the immunofluorescence staining (nuclei displayed as blue and keratin as green), a2 is surface scanning and b2 is cross‐section observation. The scaffold was surface grafted with fibronectin and implanted in porcine esophagus for 5 months (a3 and b3). Arrows in b1 indicate scaffold lumen, and in a3 and b3 referred the nude esophagus and scaffold‐implanted site.

A typical electrospinning setup includes a syringe pump, a metallic collector, and a high‐volt‐ age generator. The parameters of this electrospinning system, including process parameters (e.g., electric potential, solution flow rate, distance between the spray nozzle and collector, etc.), polymer solution properties (e.g., solvents, solution viscosity, and concentration), and ambient parameters (e.g., temperature and humidity) influence the fiber features and internal construction.

Leong et al. yielded poly(D,L‐lactide) fibers with the diameter of ∼1 μm and nanoscale pores on each fiber through the method of electrospinning combined with phase separation. Large pores between the fibers in the whole sheet were also formed. Such multiporous struc‐ ture greatly enhanced the cell‐matrix interactions and thus promoted the adhesion of porcine esophageal epithelial cells onto the fibers [56].

We have set up a programmable electrospinning system (China Patent ZL 200810062323.8) to upgrade the apparatus's versatility. Besides the basic components, i.e., high‐voltage generator and syringe pump, an electronic controller that allows manipulating the nozzle and metallic collector was automatically incorporated into the system. Two nozzles were applied in this system. They can be connected independently to two syringe pumps via silicone tube and operated under programmable monitor to spray polymer fibers indi‐ vidually, sequentially, or simultaneously. Using this upgrade system, a uniform compos‐ ite fiber sheet consisting of polymers and natural biomaterials with the diameter ranging from 1 to 600 nm was created. These composite sheets derived from proteins and polymers showed good biocompatibility and good mechanical properties. Furthermore, a PCL fiber mesh with macroscopically alignment was electrospun on this setup. The interesting dis‐ covery is that this aligned fiber was able to switch smooth muscle cells from synthetic to contractile phenotype and hopefully to maintain the biological function of the cultured muscle tissue [57].

Grafting with ECM molecules is a good way for synthetic scaffolds to improve their bioac‐ tivity. For example, poly(L‐lactide‐co‐caprolactone) (PLLC) was electrospun to form fibers. After then, they were grafted with fibronectin in order to promote epithelial cell growth [58]. According to the findings about the topographic features and protein quantifications of the basement membrane of porcine esophagus [4], an electrospun scaffold was fabricated using fibroin (extracted from pregnant silkworm originated in Zhejiang province, China) and polymer as the materials. In order to simulate the architecture of the basement membrane, proteins including collagen IV, laminin, entactin, proteoglycans (PG) extracted from porcine esophagus were coated on the above fibers, aiming at enhancing epithelium regeneration

A typical electrospinning setup includes a syringe pump, a metallic collector, and a high‐volt‐ age generator. The parameters of this electrospinning system, including process parameters (e.g., electric potential, solution flow rate, distance between the spray nozzle and collector, etc.), polymer solution properties (e.g., solvents, solution viscosity, and concentration), and ambient parameters (e.g., temperature and humidity) influence the fiber features and internal

**Figure 3.** Overview of tubular scaffold (a1) and cross‐section structure (b1). The scaffold was prepared with TIPS technique using biodegradable poly(l‐lactide‐co‐caprolactone) as the substrate material. Primary epithelial cells were cultured on the scaffold lumen for 14 days and the immunofluorescence staining (nuclei displayed as blue and keratin as green), a2 is surface scanning and b2 is cross‐section observation. The scaffold was surface grafted with fibronectin and implanted in porcine esophagus for 5 months (a3 and b3). Arrows in b1 indicate scaffold lumen, and in a3 and b3

construction.

182 Esophageal Abnormalities

referred the nude esophagus and scaffold‐implanted site.

**Figure 4.** Scaffold morphology of PCL/SF (a), scaffold coated with basement membrane proteins that were extracted from porcine esophagus (b), and cell phenotype immune‐histochemically stained with CK14 antibody (green) and nuclei with DAPI (blue) (c).

(**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‐ geal tissue engineering [59].
