**2. Materials for scaffold constitution**

surgery, radiotherapy, or/and chemotherapy, and surgically replacing with stomach, colon, small intestine, etc. did not improve greatly the survival rate. In addition, esophagus donor is too rare to get autologous/allogeneic replacement from human body. A tissue‐engineered substitute with integrated structure and function is thought to be a promising and effective alternative for treating esophageal disease, which will eliminate the need to harvest replace‐

The esophagus is a muscular canal extending from pharynx to stomach and has functions to transport food and water from mouth to stomach. There are three types of cells, i.e., strati‐ fied squamous epithelial cells, fibroblasts, and smooth/skeletal muscle cells, which constitute four layers of this tissue, namely the mucosa, submucosa, muscularis externa, and adventitia. **Figure 1** shows the sketch and histological structure of human esophagus, in which a folding lumen is observed in a resting state (**Figure 1a**). The stratified squamous epithelial cells (E) compose the lumen epithelium that serves as a barrier or protective layer against mechanical stresses produced by food bolus. The epithelial cells are supported by the underlying base‐ ment membrane (**Figure 1b**, arrows). The topography of the basement membrane is a rugged and uneven stripe that consists of interwoven fibers. The diameters of these fibers were mea‐ sured to be from 28 to 165 nm with an average of 66 ± 24 nm. The pores displayed between fibers with unequal size (**Figure 1**c). The molecular components of the basement membrane

**Figure 1.** Overview and histological structure of esophagus (a). There are four tissue layers, i.e., mucosa containing epithelium (E), lamina propria and muscularis mucosae, submucosa (SM), muscularis externa consisting of two sub‐ layers of inner circular (IC) and outer longitudinal (OL) muscle, and adventitia in esophagus organ. The stratified squamous epithelial cells (E) lined the esophagus lumen (H&E staining). Cross‐section and topography of basement

membrane were observed under transmission electron microscope (TEM) (b, c).

ment tissues from the patients' own body or other human body.

174 Esophageal Abnormalities

Since the ancient times, some allogeneic materials have been adopted for medicinal purposes. In the past decades, after the concept of tissue engineering was submitted, many natural and artificial materials were adopted to produce scaffolds. Some achievements have been obtained toward improving tissue regeneration in laboratory scaffold, serving as a temporary platform to support or promote the growth of cells or/and tissues, which is one of the key issues in the research of tissue engineering. For esophagus, a variety of natural or/and artificially synthe‐ sized materials have been investigated as scaffold substrate.

#### **2.1. Natural biomaterials**

Natural biomaterials, for example, collagen, chitosan, gelatin, decellularized extracellular matrix (ECM), etc., all of which are derived from animal sources, have been widely studied in scaffold constitution, since all these materials possesses good biocompatibility and have bio‐ specific signals cued from the molecules secreted by the resident cells. Thus, they are believed to be able to direct the *in vivo* remodeling process. These natural materials also have been used in a variety of tissue engineering applications, such as the grafts of heart, heart valve, skeletal muscle, skin, cardiovascular grafts, etc. For the research of esophageal tissue engineering, some ECMs like acellular dermal grafts, gastric acellular matrix, aortal acellular matrix grafts, and decellularized esophagus and urinary bladder submucosa had once been tested to repair esophagus in animal models [11–14]. For example, Marzaro et al. seeded porcine esophageal smooth muscle cells (SMC) on the acellular esophagus aiming at healing the defected porcine esophagus. They got results that SMCs grew on the ECM without obvious inflammation and rejection after implantation for 3 weeks [14].

The research team led by Professor Badylak pioneers the scaffold fabrication using decel‐ lularized ECM like porcine urinary bladder matrix (UBM) and small intestinal submucosa (SIS) toward *in vitro* and *in vivo* repairing of esophagus organ [15–17]. For example, they implanted the acellular UBM at the esophagus defects of female mongrel dogs, where the cir‐ cumferential endo‐mucosa/submucosa had been resected. The results showed that complete epithelialization took place on the scaffold surface at day 35, neovascularization and forma‐ tion of muscle bundles took place at day 50, and the immature nerves and Schwann cells were observed at day 91. After implantation for 230 days, neonatal esophagus with the formation of well‐organized tissue laminar and tissue motility had grown [12]. Another important applica‐ tion is skeletal muscle ECM. They decellularized skeletal muscle with enzymes and chemicals to obtain acellular ECM. This ECM was verified to contain the growth factors, glycosamino‐ glycans, and basement membrane structural proteins. Expectedly, these components greatly promoted myogenic cells' growth and proliferation *in vitro*, and also promoted the myogen‐ esis when the ECM was implanted into a rat abdominal wall. The ECM scaffold was found to degrade gradually at the implant site [16]. The xenogeneic ECM derived from porcine SIS combined with endoscopic technique was adopted to repair dysfunctional esophagus of five male patients who had esophageal adenocarcinoma, Barrett's or/and high‐grade dysplasia (HGD). After 24 months, patients restored the mature squamous epithelium and returned a normal diet without significant dysphagia. Unfortunately, among these five patients, some recurred Barrett's esophagus, mainly at the gastroesophageal junction [17]. It was the first and very important report about clinical application of tissue‐engineered esophagus in human body. A model of the human esophageal mucosa was reported recently by the MacNeil labo‐ ratory [18]. Unlike conventional 2D cell culture systems, they seeded primary human esopha‐ geal fibroblasts and epithelial cells in a porcine‐derived acellular esophageal scaffold and discovered an esophageal mucosa recapitulation after 20 days. It provided a biologic‐relevant experiment model of human esophageal mucosa.

Some literatures reported that three‐dimensional biological scaffolds made from nonau‐ tologous extracellular matrix (ECM) can act as an inductive template for tissue and organ reconstruction after the ECM was recellularized with autologous stem cells or differentiated cells. This kind of ECM/cells was tried to repair and reconstruct some complex tissues like esophagus, trachea, and skeletal muscle in animal models. Porcine SIS was once attempted to be used as the scaffold by Wei et al., on which the canine oral epithelial cells were pre‐ seeded before transplantation into animal body followed by suturing across an esophageal gap in the cervical portion (∼5 cm). Not only reepithelization but also muscle formation was discovered in the cell‐seeded SIS after implantation in animal body for 8 weeks [19]. Urita et al. used the decellularized stomach tissue to evaluate the esophageal mucosa regeneration [13]. Isch et al. implanted a commercial decellularized product, AlloDerm® (LifeCellTM), for the esophagoplasty of canine cervical esophagus. Complete epithelialization on the mem‐ brane surface was achieved after 2 weeks, without obvious anastomotic fistula or stenosis [20]. Bhrany compared the growth of rat epithelial cells on esophageal ECMs decellularized by deoxycholic acid and Triton X‐100. The results indicated that treatment with deoxycholic acid was better than Triton X‐100 treatment in epithelium regeneration [11]. Koch et al. decel‐ lularized a porcine esophagus and implanted subcutaneously into Sprague‐Dawley rats. The decellularized esophagus was shown to maintain its native matrix morphology and extracel‐ lular matrix composition [21]. Considering the findings in these literatures, we believed that the decellularized ECM is a good scaffold candidate in esophageal tissue engineering.

Proteins or/and proteoglycans derived from animal ECM are also the important materials that have been actively researched previously. Saxena et al. seeded rat esophageal epithelial cells on the collagen‐based scaffolds (OptiMaix‐3D001315). After cultured *in vitro*, cells were tested to display positive pan cytokeratin PCK‐26 which broadly recognizes the epitopes present in most human epithelial tissues [22]. Qin et al. fabricated a cross‐linked collagen‐chitosan sponge and implanted into the latissimus dorsi of nude mice after it was preseeded with fetal canine esophageal epithelial cells. Ten layers of mature epithelial cells formed after 2 weeks and the collagen‐chitosan implant degraded totally after 4 weeks [23]. Saito et al. implored the feasibility of collagen that used to be the substrate of tissue‐engineered esophagus [24]. They constituted an artificial esophagus using collagen sponge together with a latissimus dorsi muscle flap and split‐thickness skin and replaced the esophagus of rabbits. Five in 12 total experimental rabbits survived without anastomotic leakage or stenosis. The longest survival period in these rabbits was 16 days.

Although many interesting achievements about natural biomaterials have been obtained in some *in vitro* or *in vivo* experiments, problems like weak mechanical strength, fast degrada‐ tion, and source limitation of these natural biomaterials are still to be worked out.

#### **2.2. Polymeric materials**

**2.1. Natural biomaterials**

176 Esophageal Abnormalities

rejection after implantation for 3 weeks [14].

experiment model of human esophageal mucosa.

Natural biomaterials, for example, collagen, chitosan, gelatin, decellularized extracellular matrix (ECM), etc., all of which are derived from animal sources, have been widely studied in scaffold constitution, since all these materials possesses good biocompatibility and have bio‐ specific signals cued from the molecules secreted by the resident cells. Thus, they are believed to be able to direct the *in vivo* remodeling process. These natural materials also have been used in a variety of tissue engineering applications, such as the grafts of heart, heart valve, skeletal muscle, skin, cardiovascular grafts, etc. For the research of esophageal tissue engineering, some ECMs like acellular dermal grafts, gastric acellular matrix, aortal acellular matrix grafts, and decellularized esophagus and urinary bladder submucosa had once been tested to repair esophagus in animal models [11–14]. For example, Marzaro et al. seeded porcine esophageal smooth muscle cells (SMC) on the acellular esophagus aiming at healing the defected porcine esophagus. They got results that SMCs grew on the ECM without obvious inflammation and

The research team led by Professor Badylak pioneers the scaffold fabrication using decel‐ lularized ECM like porcine urinary bladder matrix (UBM) and small intestinal submucosa (SIS) toward *in vitro* and *in vivo* repairing of esophagus organ [15–17]. For example, they implanted the acellular UBM at the esophagus defects of female mongrel dogs, where the cir‐ cumferential endo‐mucosa/submucosa had been resected. The results showed that complete epithelialization took place on the scaffold surface at day 35, neovascularization and forma‐ tion of muscle bundles took place at day 50, and the immature nerves and Schwann cells were observed at day 91. After implantation for 230 days, neonatal esophagus with the formation of well‐organized tissue laminar and tissue motility had grown [12]. Another important applica‐ tion is skeletal muscle ECM. They decellularized skeletal muscle with enzymes and chemicals to obtain acellular ECM. This ECM was verified to contain the growth factors, glycosamino‐ glycans, and basement membrane structural proteins. Expectedly, these components greatly promoted myogenic cells' growth and proliferation *in vitro*, and also promoted the myogen‐ esis when the ECM was implanted into a rat abdominal wall. The ECM scaffold was found to degrade gradually at the implant site [16]. The xenogeneic ECM derived from porcine SIS combined with endoscopic technique was adopted to repair dysfunctional esophagus of five male patients who had esophageal adenocarcinoma, Barrett's or/and high‐grade dysplasia (HGD). After 24 months, patients restored the mature squamous epithelium and returned a normal diet without significant dysphagia. Unfortunately, among these five patients, some recurred Barrett's esophagus, mainly at the gastroesophageal junction [17]. It was the first and very important report about clinical application of tissue‐engineered esophagus in human body. A model of the human esophageal mucosa was reported recently by the MacNeil labo‐ ratory [18]. Unlike conventional 2D cell culture systems, they seeded primary human esopha‐ geal fibroblasts and epithelial cells in a porcine‐derived acellular esophageal scaffold and discovered an esophageal mucosa recapitulation after 20 days. It provided a biologic‐relevant

Some literatures reported that three‐dimensional biological scaffolds made from nonau‐ tologous extracellular matrix (ECM) can act as an inductive template for tissue and organ Artificial polymers have been much investigated as scaffold substrates in tissue engineering because these materials possess many attracting features, for example, good availability, low cost, and high possibility of designing and production. Polyethylene (PE) tube was the earli‐ est example to be an artificial conduit for esophageal replacement in a dog model in 1952 [25]. However, 6 in 20 experimental dogs died after the PE tube was replaced. Leakage and stricture in some dogs were discovered at the site of the PE junction. A fibrous sheath around this plastic tube was developed in all dogs, likely because of the nonbiodegradability of PE materials. Despite the failures, this study is the pioneering experiment of tissue engineering research of esophagus.

Since the concept of tissue engineering was put forward, the interests in using biocompat‐ ible and biodegradable polymers, such as polyglycolic acid (PGA), polycaprolactone (PCL), poly‐L‐lactic acid (PLLA), and their copolymers, has been increasing greatly, particularly after the regulatory approval from the U.S. Food and Drug Administration (FDA). Various kinds of biodegradable polymers have been investigated specifically for the tissue engineering of esophagus. For example, vicryl, made from PGA and collagen coating, is one type of biode‐ gradable and absorbable scaffold with good biocompatibility. It has been tested as a porcine thoracic esophageal replacement in 1991 [26]. No matter what, it failed when the material was implanted into animal body. It was because the reflux gastric fluid dissolved the grafted tube, resulting in severe mediastinitis, leakage, and stenosis. In 1998, Shinhar used vicryl mesh to repair the porcine cervical esophagus. Here, stenosis still took place, though the stump fistula disappeared [27]. Miki et al. fabricated a tube using PGA mesh as a frame, and collagen con‐ taining esophageal fibroblasts and epithelial cells as the outer and inner layer, respectively. The fibroblasts were found to be able to accelerate the proliferation and differentiation of epithelial cells due to the keratinocyte growth factor secreted by fibroblasts. After the tube was implanted into muscle flaps of athymic rats for 14 days, nonstenosis was observed in the tube's lumen, but also 20 layers of stratified epithelium were developed from histological examination [28].

One key issue of those synthetic materials used as scaffold matrices is the materials' hydro‐ phobic and biologically inert surface, which will inevitably lead to the inferior reactions between material and cells when the host cells come into contact with the scaffold surface upon implantation. Zhu et al. developed some methods to modify the surface chemistry aim‐ ing at enhancing cell‐polymer interactions. In order to graft proteins or other biomolecules onto polymer surface, a reaction of ester groups from the substrate polyesters (e.g., PLLA, PU, PCL, and their copolymers) and amino groups (‐NH2 ) of hexanediamine was firstly intro‐ duced to produce pendent amino groups on polyester surfaces through formation of amide bonds. This reaction was called as aminolysis. The density of amino group produced from the aminolysis reaction was quantified using ninhydrin method and fluorescein labeling. Second, this pendent ‐NH2 reacted with one aldehyde group (‐CHO) from glutaraldehyde (GA). Third, the other aldehyde of GA was used in covalently bond proteins or other biomol‐ ecules. Collagen, gelatin, chitosan, fibronectin, polypeptides, growth factors, etc. were thus grafted on the polymeric scaffold surface. Finally, the protein or other biomolecule‐grafted surfaces were produced. The water soluble carbodiimide (WSC) can also induce the reaction between the pendent ‐NH2 on the aminolyzed surface and –COOH of target proteins, so that the proteins were covalently bonded to the material surface as the **Scheme 1** demonstrated [29–31].The introduction of the amino groups also allows layer‐by‐layer (LBL) assembly on the polymer surface, because the aminolyzed polyester can be used as a polycationic substra‐ tum, on which polyanions can be assembled by means of electrostatic attraction. For exam‐ ple, LBL assemblies of poly(styrene sulfonate, sodium salt) (PSS)/chitosan and chondroitin sulfate (CS)/collagen were performed on the aminolyzed poly‐L‐lactide (PLLA‐NH2 ) surface (**Scheme 1**) [32, 33].

#### Tissue Engineering of Esophagus http://dx.doi.org/10.5772/intechopen.69350 179

this plastic tube was developed in all dogs, likely because of the nonbiodegradability of PE materials. Despite the failures, this study is the pioneering experiment of tissue engineering

Since the concept of tissue engineering was put forward, the interests in using biocompat‐ ible and biodegradable polymers, such as polyglycolic acid (PGA), polycaprolactone (PCL), poly‐L‐lactic acid (PLLA), and their copolymers, has been increasing greatly, particularly after the regulatory approval from the U.S. Food and Drug Administration (FDA). Various kinds of biodegradable polymers have been investigated specifically for the tissue engineering of esophagus. For example, vicryl, made from PGA and collagen coating, is one type of biode‐ gradable and absorbable scaffold with good biocompatibility. It has been tested as a porcine thoracic esophageal replacement in 1991 [26]. No matter what, it failed when the material was implanted into animal body. It was because the reflux gastric fluid dissolved the grafted tube, resulting in severe mediastinitis, leakage, and stenosis. In 1998, Shinhar used vicryl mesh to repair the porcine cervical esophagus. Here, stenosis still took place, though the stump fistula disappeared [27]. Miki et al. fabricated a tube using PGA mesh as a frame, and collagen con‐ taining esophageal fibroblasts and epithelial cells as the outer and inner layer, respectively. The fibroblasts were found to be able to accelerate the proliferation and differentiation of epithelial cells due to the keratinocyte growth factor secreted by fibroblasts. After the tube was implanted into muscle flaps of athymic rats for 14 days, nonstenosis was observed in the tube's lumen, but also 20 layers of stratified epithelium were developed from histological examination [28]. One key issue of those synthetic materials used as scaffold matrices is the materials' hydro‐ phobic and biologically inert surface, which will inevitably lead to the inferior reactions between material and cells when the host cells come into contact with the scaffold surface upon implantation. Zhu et al. developed some methods to modify the surface chemistry aim‐ ing at enhancing cell‐polymer interactions. In order to graft proteins or other biomolecules onto polymer surface, a reaction of ester groups from the substrate polyesters (e.g., PLLA,

duced to produce pendent amino groups on polyester surfaces through formation of amide bonds. This reaction was called as aminolysis. The density of amino group produced from the aminolysis reaction was quantified using ninhydrin method and fluorescein labeling.

(GA). Third, the other aldehyde of GA was used in covalently bond proteins or other biomol‐ ecules. Collagen, gelatin, chitosan, fibronectin, polypeptides, growth factors, etc. were thus grafted on the polymeric scaffold surface. Finally, the protein or other biomolecule‐grafted surfaces were produced. The water soluble carbodiimide (WSC) can also induce the reaction

the proteins were covalently bonded to the material surface as the **Scheme 1** demonstrated [29–31].The introduction of the amino groups also allows layer‐by‐layer (LBL) assembly on the polymer surface, because the aminolyzed polyester can be used as a polycationic substra‐ tum, on which polyanions can be assembled by means of electrostatic attraction. For exam‐ ple, LBL assemblies of poly(styrene sulfonate, sodium salt) (PSS)/chitosan and chondroitin

sulfate (CS)/collagen were performed on the aminolyzed poly‐L‐lactide (PLLA‐NH2

reacted with one aldehyde group (‐CHO) from glutaraldehyde

on the aminolyzed surface and –COOH of target proteins, so that

) of hexanediamine was firstly intro‐

) surface

research of esophagus.

178 Esophageal Abnormalities

PU, PCL, and their copolymers) and amino groups (‐NH2

Second, this pendent ‐NH2

between the pendent ‐NH2

(**Scheme 1**) [32, 33].

**Scheme 1.** Diagram of reactions between ester groups from synthesized polyesters and amino groups from diamine, aiming at introducing pendent amino groups onto substrate surface, through which many biomolecules containing amino or/and carboxylic groups can be bonded via crosslinking reagents or layer‐by‐layer assembly technology.

We tried another method, photo‐oxidation plus copolymerization, to modify the material surface chemistry. This method was processed under UV initiation to introduce carboxylic groups (‐COOH) onto material surface. Through these carboxylic groups, molecules like protein or other bio‐molecules (containing COOH) will be bonded onto the surface under the catalysis of 1‐ethyl‐3‐(3‐dimethylamino propyl) carbodiimide hydrochloride (EDAC). As a result, covalent immobilization of proteins onto the material surface took place [34]. The optimal conditions for each method with respect to cell functions have been elucidated by *in vitro* evaluation of endothelial cells or esophagus cells including epithelial cell, fibroblast, and smooth muscle cell [30, 35–39].

Another important issue regarding polymers is the catalyst used during synthesis. Those ester‐containing polymers are usually synthesized under catalyzing of stannum compounds [40, 41]. However, this kind of catalyst covalently links to the molecular chain of the ultimate products. We know, these stannum‐containing materials are harmful to human body when it is implanted *in vivo* as a scaffold substrate, because the bonded stannum will release and accumulate in body as the material gradually degrades. Thus, it is necessary to develop new methods to catalyze efficient polymerizations but no toxicity giving off. Stolt et al. explored the reaction of L‐lactide ring‐opening polymerization using catalysts generated from iron and acetic acid, isobutyric acid, butyric acid, trifluoroacetic acid, dichloroacetic acid, etc. They discovered that the iron acetate, iron isobutyrate, and iron trifluoroacetate were the efficient catalysts for ring‐opening reaction to yield poly(l‐lactide) (PLLA) with a molar mass (weight average molecular weight, Mw) of 150 kDa. The monomers' conversion was up to over 85% under the optimum reaction conditions [42]. After this, they produced lactic acid‐based poly(ester‐urethane) (PEU) using iron monocarboxylates as the initiators, which were prepared from the reaction between iron powder and acetic acid, trifluoroacetic acid, or isobutyric acid. These iron monocarboxylates were considered as catalysts in reactions of 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‐ rolactone, glycolic acid, etc. using ferric chloride (FeCl3 ), ethanol iron (Fe(OC2 H5 ) 3 ), iron (III) acetylacetonate (Fe(acac)3 ), or iron (II) acetylacetonate Fe(acac)2 , as the catalyst. The result was that Fe(acac)3 was the most efficient catalyst among them to yield products with high mono‐ 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 ini‐ 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 growth of porcine esophageal cells like epithelial, fibroblast, and muscle cell.
