Esophageal tissue engineering.

Review

Esophageal tissue engineering Expert Review of Medical Devices Downloaded from informahealthcare.com by Michigan University on 10/16/14 For personal use only.

Expert Rev. Med. Devices 11(2), 225–241 (2014)

Guillaume Luc1,2, Marle`ne Durand2, Denis Collet1, Fabien Guillemot3 and Laurence Bordenave*2,3 1 Department of Digestive Surgery, University Hospital Haut-Le´veˆque, Av de Magellan, 33604 Pessac cedex, France 2 Centre d’investigation Clinique innovations technologiques, PTIB University Hospital Xavier Arnozan, Av du Haut Le´veˆque, 33600 Pessac, France 3 INSERM, U1026, University Victor Segalen Bordeaux 2, 146 rue Le´o-Saignat, 33076 Bordeaux cedex, France *Author for correspondence: Tel.: +33 557 102 862 Fax: +33 557 102 869 [email protected]

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Esophageal tissue engineering is still in an early state, and ideal methods have not been developed. Since the beginning of the 20th century, advances have been made in the materials that can be used to produce an esophageal substitute. Three approaches to scaffold-based tissue engineering have yielded good results. The first development concerned non-absorbable constructs based on silicone and collagen. The need to remove the silicone tube is the main disadvantage of this material. Polymeric absorbable scaffolds have been used since the 1990s. The main polymeric material used is poly (glycolic) acid combined with collagen. The problem of stenosis remains prevalent in most studies using an absorbable construct. Finally, decellularized scaffolds have been used since 2000. The promises of this new approach are unfulfilled. Indeed, stenosis occurs when the esophageal defect is circumferential regardless of the scaffold materials. Cell supplementation can decrease the rate of stenosis, but the type(s) of cells and their roles have not been defined. Finally, esophageal tissue engineering cannot provide a functional esophageal substitute, and further development is necessary prior to conducting human clinical studies. KEYWORDS: cells seeding • esophagus • scaffolds • stenosis • tissue engineering

Clinical needs

Esophagectomy is a surgical procedure performed to treat two conditions, malignant or benign lesions. Benign lesions are those due to caustic ingestion, benign tumors and perforations. Malignant tumors are adenocarcinoma and squamous cell carcinoma. The worldwide incidence of esophagus malignancy is 481,645, causing 406,533 deaths annually [201]. This cancer is highly aggressive and is the sixth leading cause of cancer death worldwide [1]. There has been a marked increase in the incidence of adenocarcinoma of the esophagus over the past three decades [2]. The five-year survival rate remains low, with a range of 5–20% [3]. The only curative treatment for esophagus malignancy is surgery with or without chemotherapy and/or radiotherapy. The 2-year survival rate with curative treatment is 43% [4]. For the majority of patients, esophagectomy is performed because of malignancy, for which a wide local excision and additional lymphadenectomy are indicated [5]. The vagal nerves must be sacrificed bilaterally, which can induce a variety of functional abnormalities [6]. The continuity of the gastrointestinal tract is preferably restored using a whole-stomach 10.1586/17434440.2014.870470

interposition or gastric tube. However, the preparation of the stomach as an esophageal substitute is associated with substantial alterations: the blood supply is decreased by 10–20% due to the ligation of the left gastric and left gastroepiploic arteries [7]; the gastric capacity is reduced by resecting part of the corpus and fundus; the innervation is altered as a result of complete vagotomy and the organ is relocated to an intrathoracic position with the destruction of the normal antireflux barrier (FIGURE 1). The main complication after esophagectomy is anastomotic stenosis. The incidence of anastomotic stenosis remains relatively high (10–56%) [8]. A majority of patients will suffer from reflux symptoms after esophagectomy and gastroplasty [9], including heartburn, regurgitation, dysphagia, odynophagia, vomiting and aspiration. At 1 year post surgery, reflux symptoms were experienced by 50% of patients with intrathoracic anastomosis [10]. On endoscopy, severe reflux esophagitis (grade C or D according to the Los Angeles classification) was observed in 76% of the patients complaining of reflux symptoms [9]. Vagal denervation can result in chronic dysmotility of the gastric remnant and outlet dysfunction of the

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Luc, Durand, Collet, Guillemot & Bordenave

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A

B

Figure 1. Ivor-Lewis procedure. (A) First step: abdominal approach, preparation of the gastroplasty. (B) Second step: thoracic approach, ascension of the esophageal substitute. 90% of esophagectomies for esophageal cancer are doing with Ivor-Lewis technique in France.

pylorus, which may cause delayed emptying [6]. Pyloroplasty fails to prevent the delayed gastric emptying [11] but prevents the potentially lethal effects of gastric stasis [12]. The combination of biliary and acid reflux is commonly believed to play a central role in the pathogenesis of Barrett’s metaplasia in patients suffering from gastroesophageal reflux disease [6]. Franchimont et al. [13] found a 13.5% incidence of newly developed Barrett’s esophagus in the cervical stump after esophagectomy for cancer, with a median time to diagnosis of 489 days (range of 43–1172 days). Proton pump inhibition initiated soon after surgery did not appear to affect the development of Barrett’s esophagus. After esophagectomy followed by gastroplasty, many patients complain of diarrhea and dumping (-like) symptoms, with a reported incidence of 10– 50% [14]. The symptoms include early postprandial abdominal and vasomotor symptoms resulting from osmotic fluid shifts and the release of vasoactive neurotransmitters as well as late symptoms secondary to reactive hypoglycemia [15], including diarrhea, abdominal cramps, nausea, dizziness, postprandial sweating and hypotension. Apart from substantial perioperative morbidity and even mortality, surgical therapy for esophageal carcinoma can be accompanied by severe long-term functional disturbances that have a large impact on quality of life. However, studies have suggested that quality of life was restored in patients (disease-free survival) within 9 months of the operation [16]. The results were different if the patients were treated by multimodality therapy [17]. The gastric pull-up approach has been fraught with common postoperative complications, such as anastomotic leakages, strictures, mediastinisis, diarrhea, dumping (-like) symptoms and reflux problems [18]. The use of gastrointestinal segments to bridge the gap has been considered the gold standard approach, although postoperative complications can lead to morbidity and mortality [19]. Allografts and xenografts comprising acellular matrices are alternatives to gastrointestinal segments, but their immunogenicity and 226

possible disease transmission limit their widespread use. Thus, tissue engineering and regenerative medicine are being utilized to develop alternative strategies to create imitations of the natural tissue that could be used as artificial grafts for esophageal regeneration [18,19]. Properties & characteristics of the esophagus Histological characteristics of the esophagus

In addition to bridging the esophageal gap after its surgical removal, a successful esophageal replacement must also be able to reestablish the native peristaltic movements necessary for proper food transit. This function requires the esophageal scaffold to reproduce the important structural features of the principle tissue layers involved in peristaltic motion, namely, the mucosa, submucosa and muscularis externa (FIGURE 2) [20]. The mucosa is composed of three different layers. The innermost layer is a stratified, non-keratinized squamous epithelium with a basement membrane. The basement membrane separates the epithelium from the remainder of the esophageal wall. The lamina propria is a thin layer of connective tissue containing extracellular matrix (ECM), fibers (collagen III and elastin), vessels (capillaries and lymphatic), sensory nerve endings and scattered areas of lymphoid tissue [21]. External to the lamina propria is the muscularis mucosae, which is a layer consisting of smooth muscle and elastic fibers. The smooth muscle fibers in the muscularis mucosae are arranged in two concentric layers with different orientations: an inner circular (IC) layer that encompasses the lumen and an outer longitudinal (OL) layer parallel to the long axis of the tract. The submucosa is dense connective tissue joining the mucosa to the muscularis externa. It is rich in elastin and collagen with a complex distribution pattern [22]. The type III collagen fibrils within the submucosa layer have a circumferential orientation. The orientation of the collagen and elastin fibrils in this layer contributes to its circumferential distensibility without the loss Expert Rev. Med. Devices 11(2), (2014)


Esophageal tissue engineering

Review

of longitudinal strength [23,24]. Mucoussecreting glands are present in this layer. The submucosal layer also contains Meissner’s plexus, which regulates secretion and the peristaltic contractions of MM the muscularis mucosae, blood vessels OL and lymphatic vessels [18]. The muscularis externa forms the musE cular wall and can be subdivided into two distinct layers based on their orientation. The cells of the IC muscle layer IC SM lying adjacent to the submucosa are oriented circumferential to the esophageal 450 µm axis, whereas those in the second, OL layer are aligned parallel to the esophaFigure 2. Cross-section showing the tissue organization in the porcine esophageal lumen [25]. Between the two muscle gus. The mucosa, consisting of epithelium (E) and the muscularis mucosa (MM); the submucosa (SM) and muscularis externa, consisting of an inner circular (IC) and outer layers are the nerve fibers and ganglia longitudinal (OL) muscle layer. that control the muscle contractions (myenteric or Auerbach’s nerve plexus). The muscle cell types change along the length of the muscularis determined to be 1.2 MPa, and the distortion reached 57%. externa. Both skeletal and smooth muscle cells are present in The esophagi of the rat [29], guinea pig [30], porcine [28] and the mid-esophagus, and only smooth muscle cells are in the human [27,31] exhibit similar mechanical behaviors even though different values have been obtained for their mechanical propinferior portion. erties. These models display significantly higher ultimate strength and elastic moduli in the mucosal-submucosal layer Mechanical properties of the esophagus The esophagus is a highly elastic tissue exhibiting anisotropic than in the muscle layer. This non-linear mechanical feature of behavior [24,26]. Vanags et al. [27] measured the mechanical char- the mucosal–submucosal layer could be essential to promoting acteristics of 104 human esophagi. Esophageal stress, strain, deformation and flattening of the folds during the passage of a ultimate strain deformation energy and the tangential modulus food bolus [28]. Natali et al. have shown that the anisotropic of elasticity were evaluated using esophageal samples (4 behavior of the submucosal–mucosal layer is due to the double 25 mm). Values were obtained in the longitudinal and circum- crisscrossing helix configuration of the collagen fibers [24]. The ferential directions and subdivided in four groups according to orthotropic behavior of the muscularis externa is due to the age and region of the esophagi (cervical, thoracic and abdomi- orthogonal orientation of the muscular fibers. The mechanical nal). For example, in age group I (19–44 years), the average behavior of the esophagus is dependent on the presence of elasultimate strain for the cervical region was 2.80 MPa, which is tin and collagen fibers. Their spatial arrangement is important 42.1% greater than that of the thoracic region of this organ for the biomechanical properties [32]. Elastin assumes the form and 54.7% greater than that of the abdominal part of the of wavy fibers with a principally longitudinal alignment in esophagus in this age group (p < 0.05). The esophageal stress, both the submucosa and adventitia but has a less organized strain, ultimate strain deformation energy and tangential modu- structure and is less abundant in the septa of the muscle layer. lus of elasticity values at 40% relative deformation of the nor- The adventitial collagen fibers have a wavy appearance and folmal wall in both the longitudinal and circumferential low a longitudinal course, and the same behavior is apparent directions were greatest in the cervical part of this organ. but less marked for the collagen in the submucosa and septa. Vanags et al. [27] reported that the human esophagus is stronger Collagen occupies a larger area than elastin in both layers and longitudinally than radially, with average ultimate strengths of directions [32]. The mechanical properties and spatial arrangement of the 2.19 and 1.41 MPa and average elastic moduli of 2.30 and 1.44 MPa, respectively. The opposite trend was observed for ECM should be considered. Future scaffold constructs will be extensibility. Esophagus can be stretched circumferentially by successful only if their mechanical properties and spatial 50% under pressure in the range of 3–5 kPa that is exerted by arrangement are similar to those of the esophageal ECM [33]. a food bolus of the normal size of 10 mm [28–31]. The intraluminal pressure for rupture was determined to be 55 kPa, Tissue engineering whereas it is 32 kPa if esophagitis was present [27]. Tissue engineering can be defined as the application of biologiEgorov et al. studied the biomechanical properties of digestive cal, chemical and physical techniques that follow equipment segments, including the esophagus [29]. These researchers deter- manufacturing principles related to design, research, purchasing mined the longitudinal strain but did not evaluate the tangen- and control, leading to the construction and commissioning of tial modulus of elasticity. 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regeneration of living tissue through the use of biomaterials, cells and/or factors. Tissue engineering is an alternative to replacing the esophagus with other gastrointestinal segments (stomach, jejunum and colon) [18,19]. Various skills are required for tissue engineering, including those of physics, mathematics, computer science, biology, medicine and robotics. The main objective of tissue engineering is the development of replacement organs, but it can also involve the development of two- or three-dimensional designs for biological studies. All of the methods are aimed at reproducing an artificial tissue-specific microenvironment using cells, growth factors and an organic or inorganic matrix [34]. The typical stages of tissue engineering are based on the scaffold and involve first removing cells from a patient and culturing them, potentially with a differentiation step. Then, the cells are seeded onto a non-degradable (synthetic) or biodegradable (natural or synthetic) scaffold material with properties that depend on the tissue to be reconstructed. Culturing of the cells on the scaffold is preferably performed under dynamic conditions (bioreactor) to facilitate the penetration of nutrients and gas exchange into the biomaterial [35]. At this stage, growth factors and differentiation factors can be used to promote tissue growth. Finally, after a sufficient period of maturation, the tissue can be implanted into the patient. The term ‘tissue engineering’ is used because tissue engineering plays a central role in the construction of replacement tissue based on a scaffold [36]. Knowledge of the histological characteristics and mechanical properties of the esophagus is necessary for envisaging esophageal tissue engineering. Different types of scaffold & the tissue response to esophageal replacement

A scaffold is a natural or synthetic complex that is biocompatible with cells. Its morphology, geometry, thickness and porosity (pore size) are known to affect cell adhesion, proliferation, tissue organization, angiogenesis and the formation of the ECM [20,21]. The porosity of the scaffold determines the degree to which fibrosis and angiogenesis occur [18]. The esophageal scaffold must be biocompatible, biodegradable and resistant to the stomach acid that is naturally present in the esophagus [37]. This structure must not cause infection, inflammation or degradation and must be compatible with cell regeneration [18]. Until cell regeneration occurs, the scaffold should be the functional substitute for the esophagus. This structure must have the mechanical and suturability properties for adaptation to the forces exerted during the passage of a food bolus. The structure’s viscoelasticity should be sufficient for it to withstand the contraction–relaxation phases of peristalsis. Several absorbable and non-absorbable grafts made of natural or synthetic materials are available for replacing the esophagus [33]. To date, a number of scaffolds have been investigated as possible replacements for the esophagus [18,19]. Non-absorbable constructs, absorbable constructs and decellularized matrices (DMs) are the most commonly used scaffolds for esophageal reconstruction at present. 228

Non-absorbable scaffolds

Various types of esophageal intubation techniques have been in use for over 165 years in attempts to palliate the symptoms of esophageal carcinoma. In 1845, there were reports of the use of decalcified ivory tubes. Similar attempts were made in the following years, and in 1887, Symonds attempted to use prostheses [38]. In 1914, Guisez introduced Pezzer-like catheters under direct visualization [39]. In 1927, Souttar reported 100 cases of using a rubber tube incorporating a ‘German silver coil’, including 18 post-mortem reports [40]. In 1954, Coyas inserted a plastic tube under direct visualization [41]. In the same year, Kropff reported on the treatment of a cervical esophagotomy by inserting a polyethylene tube with a built-in funnel at the proximal end [42]. In 1907, Bircher was the first person to report conducting an esophageal substitution using a tube made of skin [43]. In 1922, Neuhof and Ziegler used a patch of a non-absorbable material that had undergone prior granulation [44]. In the 1950s, Berman reported on a series of esophageal replacements using plastic tubes in dogs. The prosthesis used by Berman was a plastic tube with a cuff. A nylon mesh was applied to cover the cuff and the ends of the esophagus to overcome the leakage problem by permitting fibrous infiltration and firm union at the ends around the cuffs. Both methacrylate and polyethylene tubes were tested. Eating did not present a problem, and necrosis at the anastomosis occurred in only one dog [45]. In 1963, Fryfogle et al. studied replacing the middle third of the esophagus with a silicone rubber-Dacron prosthesis [46]. The proximal anastomosis became structured in all of the dogs. In this article, the same team reported the case of a 74-year-old man who was admitted for progressive dysphagia. A right thoracotomy was performed, and 15 cm of the esophagus was resected. A molded silicone rubber-Dacron graft was inserted. Six weeks postoperative, the patient was alive and had gained eight pounds [46]. In 1965, Lister et al. [47] experimented with implanting a polyethylene mesh (MarlexÒ) in 41 dogs. Two groups were compared: the first group received only the Marlex mesh, and the second group received the Marlex mesh surrounded by collagen or an anterior rectus sheath. The survival rate of group 1 was 25%, that of group 2 with collagen was 28%, and that of group 2 with an anterior rectus sheath was 70%. Stenosis occurred in the instances in which the prosthetic stent became displaced prior to 3 months after the esophageal substitution. In 1983, Fukushima et al. were the first to use a silicone tube surrounded by a Dacron ‘mesh’ [48]. These tubes were implanted in 16 dogs. The survival rate was 44% at 1 year and 25% at 6 years. They noted regeneration of the submucosa and mucosa in contact with the anastomoses. However, the central part of the tube consisted of fibrotic tissue. There were no glands or muscle tissue. This major study demonstrated that non-absorbable material does not allow for tissue growth similar to that of native tissue. The development of a dual-layer tubular structure composed of collagen and silicone appeared to be a good alternative in the 1990s. In 1993, Takimoto et al. reported the long-term results of a tubular structure composed Expert Rev. Med. Devices 11(2), (2014)


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Table 1. Esophageal tissue engineering based on the use of non-absorbable materials. Study (year)

Material

Length (mm)

Animal model

Berman (1952)

Methacrylate/polyethylene/nylon

50–90

Dog

[45]

Fryfogle et al. (1963)

Silicone/dacron

60–130

Dog

[46]

Lister et al. (1965)

Polyethylene

50

Dog

[47]

Fukushima et al. (1983)

Silicon/dacron

Dog

[48]

Ike et al. (1989)

Silicon/collagen

50

Dog

[94]

Natsume et al. (1990)

Silicon/collagen

50

Dog

[53]


Silicon/collagen

50

Dog

[54]

Takimoto et al. (1993)

Silicon/collagen

50

Dog

[49]


Silicon/collagen

50

Dog

[50]


Silicon/collagen

100

Dog

[51]


Silicon/collagen

50

Dog

[52]

Yamamoto et al. (1999)

Silicon/collagen

50

Dog

[92]


Silicon/collagen

50

Dog

[93]

Liang et al. (2009)

Silicon/nitinol

80

Pig

[55]

Liang et al. (2010)

Silicon/nitinol

80

Pig

[56]

of an outer layer of collagen and an inner layer of silicon that was implanted in dogs [49]. The inner layer that acted as a stent was removed after 4 weeks, thus limiting the formation of anastomotic strictures. Depending on the time elapsed between the introduction of the tube and removal of the stent, Takimoto et al. observed tissue regeneration similar to that in native esophageal tissue [50]. In 1995, the same team proposed utilizing the same strategy with an esophagus longer than that used previously (10 vs 5 cm), and the results were encouraging [51]. In 1998, the same group published data suggesting the potential to obtain a neo-esophagus consisting of a stratified epithelium with a striated muscle tissue arranged as an IC layer and OL layer [52]. In 1990 and 1993, Natsume et al. reported the results of the implementation of dual-layer tubes similar to those of Takimoto et al. but containing autologous epithelial cells of oral origin [53,54]. Finally, Liang et al. used a doublelayered tube but replaced collagen with nitinol and added polyester connecting rings [55,56]. Stratified squamous epithelium was observed only near the anastomosis within 1–2 months [55]. However, the rate of stenosis in the experimental group was 60%. The lack of biodegradation and long-term biocompatibility that result in stenosis, anastomotic leaks and low tissue growth requires the replacement of these materials by natural or synthetic absorbable material. TABLE 1 summarizes the various publications on esophageal tissue engineering based on the use of non-absorbable materials. Polymeric absorbable scaffolds

Polymeric scaffolds offer advantages over non-absorbable scaffolds in terms of biomechanics, cell proliferation and neoangiogenesis [18]. Synthetic polymers, such as poly (Linformahealthcare.com

Ref.

lactic acid) (PLLA), poly (lactide-co-glycolide) (PLGA, 75/ 25, 50/50), poly (caprolactone) (PCL), PCL/PLLLA, poly (L-lactide-co-caprolactone) (PLLC) and poly (glycolic acid) (PGA) have been used to develop an esophageal substitute. However, their mechanical properties and porosity are affected by their degradation. The biocompatible degradation of the scaffold material must proceed appropriately and in synchrony with the tissue formation [57]. Zhu and Ong demonstrated the biocompatibility of PCL with or without adherent epithelial cells [58]. They modified the PCL with modified collagen IV associated with 1,6-hexane diamine and glutaraldehyde and obtained better cell adhesion and increased cell proliferation, as confirmed by scanning electron microscopy (SEM). Finally, DNA quantification indicated that the PLC-collagen was better than the controls (PLC alone and polystyrene). The same group [59] also produced PLLC nanofibers by electrospinning and modified them using fibronectin. The elasticity modulus of these nanofibers was proportional to the fragility of the modified scaffold. Moreover, SEM analysis of the scaffold confirmed the improved adhesion, proliferation and cohesion of cells in the presence of fibronectin (vs the control of PLLC alone). Finally, in 2006, Zhu et al. compared the behavior of three esophageal cell lines (epithelial cells, smooth muscle cells and fibroblasts) seeded on PLLC alone, PLLC-collagen or PLLC-fibronectin [60]. Cell adhesion and proliferation were better on the modified PLLC due to the presence of R-arginine, L-glycine, D-aspartic acid (RGD) motifs in collagen and fibronectin. PLLA membranes are produced using chemical methods (solvents). They can be modified with collagen via covalent 229


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bonds using glutaraldehyde or genipin. Culturing smooth muscle cells on modified or unmodified PLLA was evaluated. The adhesion and proliferation of smooth muscle cells on the PLLA membranes were improved with collagen association. These results demonstrated that the presence of the RGD motif that is in the ECM is a key factor in tissue engineering [61]. In 2005, Zhu et al. produced collagen-grafted poly (DL-lactideco-glycolide) membranes [62]. PLGA was aminolyzed using 1,8-diaminooctane, followed by collagen immobilization using glutaraldehyde. The effect of the grafted collagen on cell behavior was investigating by comparing modified and unmodified PLGA. Unmodified PLGA was a less favorable substrate for the proliferation of esophageal smooth muscle cells. The porosity of synthetic scaffolds affects the adhesion, proliferation and connections of cells [37]. Beckstead et al. developed scaffolds with different porosities composed of PLLA, PCL or PGLA (75/25 or 50/50) [63]. They studied whether such parameters as pore size, calcium concentration and the presence of proteins (collagen I and IV, fibronectin, laminin and osteopontin) affected cell adhesion and proliferation (of epithelial cells from the rat esophagus). They compared their results to those obtained using a control (AllodermÒ, decellularized cross-linked human dermis). Low calcium concentrations allowed for increased cell growth and differentiation (Ca2+ < 0.1 mm). Pore size affected the architectural organization of the epithelial cells. Large pores allowed for better organization of a stratified epithelium. Adhesion was promoted by the presence of collagen I > collagen IV > fibronectin > laminin > osteopontin. Finally, the Alloderm scaffold (without modification) offered a more favorable architecture for the organization of the epithelium. PGA fiber-based scaffolds have been developed in association with cellular units. These units consisted of a mesenchymal nucleus surrounded by epithelial cells. The scaffolds were implanted in rats to determine whether a neo-esophagus similar to the native esophagus would grow. However, when a circumferential section of the esophagus was replaced, stenosis occurred at the upper anastomosis, whereas the tissueengineered esophagus was dilated at the lower anastomosis [64]. Nakase et al. developed two types of scaffold to provide a circumferential replacement of the intrathoracic esophagus in a canine model [65]. The first type, which lacked keratinocytes and fibroblasts (KF-), consisted of PGA associated with a sheet of amniotic membrane to which smooth muscle tissue was added. The second type (KF+) was similar to the previous one except for the addition of autologous keratinocytes and fibroblasts. The scaffolds were wrapped around a polypropylene tube that was implanted in the omentum. The KF- scaffold led to stenosis in less than 1 week after implantation, but this complication did not occur in the KF+ group. Sato et al. [66] constructed a graft of PGA-collagen seeded with esophageal epithelial cells. The graft was implanted in the latissimus dorsal muscle. Twenty days after implantation, a basement membrane appeared between the PGA graft and epithelial cells. Miki et al. developed an 230

artificial esophageal epithelium that was 20 layers thick [67]. They seeded human esophageal epithelial cells and fibroblasts onto PGA membranes. Fourteen days after transplantation in muscle flaps of athymic rats, histology revealed 20 layers of stratification in the epithelium. PGA-collagen was tested after circular myotomy in piglets [68]. The amuscular site with the collagen sponge scaffold had the same appearance as the intact esophagus. Shinhar et al. evaluated circumferential transplants of collagen-coated Vicryl meshÒ (Polyglactin 910) in dogs. Complete recovery of the esophagus was observed 6 months after implantation [69]. Jansen et al. compared polyvinylidene fluoride (PVDF) and Vicryl mesh as esophageal replacements in 10 rabbits. The histological and clinical results for PVDF were better than those for Vicryl [70]. Saxena et al. investigated two types of scaffold. The first was OptimaixÒ (collagen scaffold, Matricel GmbH, Herzogenrath, Germany) that was seeded with rat esophageal epithelial cells [71]. They engineered sheets of esophageal epithelial cells and demonstrated their viability on this collagen scaffold for an 8-week period. The same team employed the same approach using aortic smooth muscle cells and esophageal epithelial cells [72]. They developed a hybrid approach of assembling individual tissue components in vitro using BMMÒ-coated scaffolds and later assembled them to form a composite tissue. In 2010, Saxena and collaborators [73] demonstrated the consequence of using a subpopulation of epithelial cells from the ovine esophagus. The CK-26(+) subpopulation was important for generating an optimal construct. The problem of stenosis is remains prevalent in most studies using an absorbable construct [64,70]. Stenosis may be associated with scaffold degradation, which could result in the loss of mechanical stability at different phases of development and lead to anastomotic leakages [18]. According to the results of Isch et al. [74], who had studied decellularized cross-linked human dermis (Alloderm), natural decellularized scaffolds may yield better results than polymeric scaffolds [63]. TABLE 2 summarizes the various publications on esophageal IT based on a polymeric scaffold. Natural scaffolds: decellularized matrices

DMs contain a number of components of the ECM and are rich in collagen, elastin, fibronectin, laminin and growth factors. DMs mimic the biological and mechanical functions of the native ECM and have a three-dimensional architecture associated with a microenvironment that is conductive to cell growth, proliferation and orientation [75,76]. DMs are widely used in vascular tissue engineering, heart valve, skin and urological settings [18,19]. Recently, functional kidneys and lungs have been created based on the decellularization principle [77,78]. DMs are prepared using physical and chemical methods (FIGURE 3) [79]. The main chemical cell removal techniques use sodium dodecyl sulfate cell, octylphenoxypolyethoxyethanol (Triton X-100) or deoxycholic acid (DEOX) and trypsinization. Ozeki et al. compared these methods and concluded Expert Rev. Med. Devices 11(2), (2014)


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Table 2. Tissue engineering studies using resorbable polymeric scaffolds. Study (year)

Scaffold type

Animal

Cells

Sato et al. (1994)

PGA-collagen

Rats

Human esophageal epithelial cells

[66]

Shinhar et al. (1998)

Vicryl mesh-collagen

Dogs

Acellular

[69]

Miki et al. (1999)

PGA-collagen

Rats

Epithelial cells/fibroblasts

[67]

Komuro et al. (2002)

PGA-collagen

Pigs

Acellular

[68]

Griskchiet et al. (2003)

PGA-collagen

Rats

Epithelial cells + mesenchymatous nuclei

[64]

Rabbits

Acellular

[70]


Jansen et al. (2004)

Ò

Vicryl mesh /PVDF Ò

Ref.

Beckstead et al. (2005)

PGA/PLLA/PLGA/PCL/Alloderm

In vitro

Epithelial cells

[63]

Zhu et al. (2005)

PLGA-collagen

In vitro

Esophageal smooth muscle cells

[62]

Zhu et al. (2006)

PLLC-collagen

In vitro

Epithelial cells/fibroblasts/smooth muscle cells

[60]

Zhu et al. (2007)

PLLA-collagen

In vitro

Smooth muscle cells

[61]

Zhu et al. (2007)

PLLC-fibronectin

In vitro

Epithelial cells

[59]

Nakase et al. (2008)

PGA-amniotic membrane

Dog

Smooth muscle cells/fibroblasts/keratinocytes

[65]

Zhu et al. (2009)

PCL-collagen

In vitro

Epithelial cells

[58]

Saxena et al. (2009) Saxena et al. (2009) Kofler et al. (2010)

Ò

Ò

In vitro

Epithelial cells/smooth muscle cells

[71]

Ò

In vitro

Epithelial cells

[72]

Ò

In vitro

Epithelial cells

[73]

Optimaix -BMM Optimaix

Optimaix

BMMÒ: Matrigel; In vitro: No preclinical studies; OptimaixÒ: Collagen scaffold (Matricel GmbH, Herzogenrath, Germany); PCL: Polycaprolactone; PGA: Poly (glycolic) acid; PLGA: Poly (lactic-co-glycolic) acid; PLLA: Poly (L-lactide) acid; PLLC: Poly (L-lactide-co-6-caprolactone); PVDF: Polyvinylidene fluoride.

that DEOX was superior to Triton X-100. The latter alters the texture of the fabric, whereas it is preserved by DEOX [80]. Decellularization reduces inflammation and calcification of the scaffold after implantation because the content of the major histocompatibility complex is reduced during decellularization [80,81]. The DMs used in esophageal tissue engineering are derived from tissues, such as the bladder, esophagus, small intestine, stomach, dermis and aorta [18]. In 2001, Isch et al. used a decellularized human dermis (Alloderm) patch to repair a 2 1 cm esophageal defect in a canine model [74]. All of the dogs survived without a fistula, stenosis, infection or dysphagia, as shown by pharyngoesophageal transit. Histological analysis of the esophagus indicated re-epithelialization associated with neovascularization of the substituted site 1 month after surgery. In 2006, Bhrany et al. developed an esophageal DM containing ECM protein (the presence of collagen, elastin, laminin and fibronectin was confirmed by histology) and seeded with rat epithelial cells [82]. They observed the presence of a stratified epithelium (five to seven cell layers) after 11 days. The DM did induce an immune response. The same team subsequently stabilized their scaffolds by ‘cross-linking’ using glutaraldehyde or genipin and obtained satisfactory mechanical strength. Glutaraldehyde engendered cellular toxicity that did not occur with genipin [83]. This esophageal DM also allowed for the growth of smooth muscle tissue. Culturing autologous SMCs associated with the esophageal DM allowed for smooth muscle fascicles with a informahealthcare.com

minimal inflammatory response to be obtained. The formation of muscle layers was attributed to the secretion of growth factors by the autologous SMCs [84]. The use of a gastric DM (GDM) yielded satisfactory results in the replacement of the bladder and small intestine [18]. GDM patches implanted in the abdominal esophagus of rats allowed for good mucosal regeneration, as confirmed by bromodeoxyuridine staining. A keratinized epithelium was fully formed 2 weeks after implantation. There was no stenosis or dilatation [85]. Badylak et al. demonstrated the potential for using a bladderderived DM. When associated with smooth muscle tissue, this DM could be used to replace the long esophageal segment [86]. The use of the DM or bladder muscle tissue alone engendered

Figure 3. Dynamic and chemical decellularization of porcine esophagus using acid deoxycholic.

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A

B

500 µm

Figure 4. Masson’s trichrome staining of normal and acellular porcine esophagus. (A) Cells are present in the 3 layers before decellularization. (B) Masson’s Trichrome staining of decellularized porcine esophagus. No cells are present; architecture of the 3 layers is conserved.

scar tissue that was responsible for stenosis. The combination of DM and muscle tissue allowed for regeneration similar to that of the native esophagus. Using decellularized small intestinal submucosa (SIS) as a DM offered encouraging results. Lopes et al. replaced a semi-circular esophageal defect in rats using a lyophilized or fresh SIS. Both forms of SIS allowed for proper healing and the formation of a normal esophagus [87]. The inflammatory response was moderate 8 weeks after implantation. Wei et al. developed an SIS patch with associated epithelial cells. The esophageal defect created in a canine model was replaced by a SIS patch with or without epithelial cells. Healing was satisfactory in both groups, but complete reepithelialization occurred only when the patch included epithelial cells [88]. Gaujoux et al. interposed an aortic allograft after a circumferential resection of the cervical esophagus in pigs [89]. No immunosuppressive treatment was given. Maintaining a lumen through the graft area using a stent was necessary for 6 months post-surgery, and the mortality rate during the first month was 33%. Kajitani et al. interposed a porcine aortic allograft that was decellularized using Triton X-100 in a defect (thoracic esophagus in pig) created by excising half of the circumference [90]. Histology of the specimen after 7 weeks indicated a completely healed esophageal wall with a regenerated mucosa and submucosa. The muscular layer had also regenerated, although not completely. Badylak et al. [91] studied DM scaffolds derived from either the SIS or urinary bladder submucosa (UBS). Acellular DM patch grafts derived from either SIS or UBS served as resorbable constructive scaffolds for the repair of partial circumference esophageal defects in a dog model, but stenosis occurred when full circumference tube grafts were attempted. A decellularized esophageal matrix is the best choice for a scaffold. It avoids immunogenic phenomena while preserving the intrinsic characteristics of native tissue [18,19], offers the potential for autologous cellularization and can be prepared in 232

a reproducible manner (FIGURE 4). TABLE 3 summarizes the DMs used in the manufacture of bioscaffolds. Clinical outcomes in animal models

We focused on preclinical studies of in vivo and in situ esophageal replacement with tissue-engineered substitutes (including esophageal replacement in animal models, excluding subcutaneous implantation). The species used for the esophageal replacement were dogs [46–54,65,74,86,88,91–94], rats [64,85,87], pig [55,56,68,84,89,90], rabbits [70] and goats [95]. In the three major species (rat, dog and pig), histological characteristics are similar with human esophagus. Esophagus is composed of three principle tissue layers (mucosa, submucosa and muscularis externa). Some differences are observed in species (TABLE 4). The defects were circumferential or only patches. Stenosis developed more frequently when the defect was more than half circumferential and at all thicknesses [45,85,86,91]. Stenosis was the most important complication. In several studies, the authors employed a stent to prevent stenosis and fistula [48–54,92–94]. However, the stent (silicone or nitinol) must be removed 2–6 weeks after implantation. In one study [51], two animals died during the procedure to remove the stent. Esophageal stenting is already used in clinical practice to cover a post-esophagectomy fistula [96] but never to prevent stricture formation. All types of scaffold have been tested in vivo. A nonabsorbable scaffold has limited use in future clinical studies because it must be removed in an endoscopic procedure. Silicone was the scaffold employed most often in association with collagen [49–54,93,94]. Takimoto et al. demonstrated the relationship among the stenting period, regeneration of esophageal tissue and stricture formation. A silicone stent must be left for more than 4 weeks after implantation [50]. Watanabe et al. studied the peristaltic movement of a neo-esophagus composed of Goretex wrapped in nickel-titanium [95]. There was no follow-up in this study, but the neo-esophagus was simulated Expert Rev. Med. Devices 11(2), (2014)


Review


Table 3. Decellularized matrices used in manufactured bioscaffolds. Study (year)

Bioscaffold

Animal

Cells

Ref.

Badylak et al. (2000)

Pig UBM and SIS

Dog

Acellular

[91]

Kajitani et al. (2001)

Pig aorta

Pig

Acellular

[90]

Isch et al. (2001)

AllodermÒ

Dog

Acellular

[74]


Pig UBM

Dog

Striated muscle cells

[86]

Lopes et al. (2006)

Pig SIS

Rat

Acellular

[87]

Ozeki et al. (2006)

Rat EAM

In vitro

Epithelial cells

[80]

Bhrany et al. (2006)

Rat EAM

Rat (subcutaneous)

Epithelial cells

[82]

Marzaro et al. (2006)

Pig EAM

Pig

Smooth muscle cells

[84]

Urita et al. (2007)

Rat GAM

Rat

Acellular

[85]

Bhrany et al. (2008)

Rat EAM

Rat (subcutaneous)

Epithelial cells

[83]

Wei et al. (2009)

Pig SIS

Dog

Epithelial cells

[88]

Gaujoux et al. (2010)

Pig aorta

Pig

Acellular

[89]

Tan et al. (2013)

Pig SIS

Dog

BMSCs

[99]

AllodermÒ: Decellularized human dermis; BMSC: Bone marrow stem cell; EAM: Esophageal acellular matrix; GAM: Gastric acellular matrix; SIS: Small intestinal submucosa; UBM: Urinary bladder matrix.

and produced esophageal movement. However, the substitute was a Goretex prosthesis that cannot substitute for the esophagus for a long period. Polymeric scaffolds were not often employed in preclinical studies [64,65,68,70]. PGA and PVDF

scaffolds were tested in vivo. Jansen et al. obtained mucosa and muscularis externa using a PVDF substitute. There were no complications in the PVDF group compared with the Vicryl group, but the substitute concerned only a patch of the

Table 4. Histological and mechanical characteristics in animal models. Animals Dog

Characteristics

[108]

Histologic (3 layers)

Mucosa: stratified squamous non-keratinized epithelium. Submucosa: mucous gland present throughout the entire length of esophagus Muscularis externa: skeletal muscle

Mechanic

300–400 mmHg

Differences

Mucosa: muscularis mucosae is absent in cranial part, and interrupted in middle part Muscularis externa: skeletal muscle

Pig (no published personal data) Histologic (3 layers)

Mucosa: stratified squamous keratinized epithelium. Submucosa: mucous gland present in the cranial middle part of esophagus Muscularis externa: skeletal muscle in cranial part, skeletal and smooth muscle in middle part, smooth muscle in distal part

Mechanic

600–800 mmHg

Differences

Mucosa: muscularis mucosae is absent in cranial part

Rat

[109,110]

Histologic

Mucosa: stratified squamous non-keratinized epithelium. Muscularis externa: skeletal muscle in cranial part, skeletal and smooth muscle in middle part, smooth muscle in distal part

Mechanic

5–15 mmHg Mucosa: no muscularis mucosae

Differences

Submucosa: no gland


233

234

Dog (8)

Dog (5)

Dog (19)

Dog (25)

Dog (29)

Dog (7)

Dog (43)

Dog (9)

Ike et al. (1989)








Goretex Nickel-titanium

Silicone Collagen

Silicone Collagen

Silicone Collagen

Silicone Collagen

Silicone Collagen

Silicone Collagen

Silicone Collagen

Silicone Collagen

Silicone Collagen

Silicone Dacron

Silicone Dacron

Methacrylate Polyethylene

Substitute

Cervical Circumferential (200)

Thorax Circumferential (50)









Cervical circumferential

Thorax Circumferential (60–130)

Abdominal Circumferential (70–90)

Localization Defect characteristics (length in mm)

No

Omentum

No

No

No

No

No

Epithelial layer

Epithelial cells (oral mucosal cells of dogs/ autologous)

No

No

No

No

Cells (source)

0

36

24

4

12

12

24

5 5

7 3

27 0

2 2

15 0

11 8

0 0

0 0

0.5

1

3 5

7 0

8 4

13 6

Morbidity Mortality

1

72

5

17

Follow-up (months)

Peristaltism

Mucosa (no in midregion) Muscularis mucosae Muscularis externa (no mature)

Mucosa Muscularis externa except in midregion

Mucosa Submucosa Muscularis externa



Mucosa

Mucosa

Mucosa

Mucosa

Mucosa Submucosa

Scar tissue

Scar tissue

Histological results

AllodermÒ: Decellularized human dermis; BMSC: Bone marrow stem cell; BMMÒ: Matrigel; EAM: Esophageal acellular matrix; GAM: Gastric acellular matrix; PCL: polycaprolactone; PGA: Poly (glycolic) acid; PLLA: Poly (L-lactide) acid; PLLC: Poly (L-lactide-co-6-caprolactone); PLGA: Poly (lactic-co-glycolic) acid; PVDF: Polyvinylidene fluoride; SIS: Small intestinal submucosa; UBS: Urinary bladder submucosa.

Goat (1)

Dog (16)

Fukushima et al. (1983)

Watanabe et al. (2005)

Dog (8)

Fryfogle et al. (1963)

Dog (14)

Dog (20)

Berman (1952)


Animals (n)

Study (year)

Table 5. In vivo and in situ implantation studies of esophageal replacement.


[95]

[93]

[92]

[52]

[51]

[50]

[49]

[54]

[53]

[94]

[48]

[46]

[45]

Ref.

Review Luc, Durand, Collet, Guillemot & Bordenave

Expert Rev. Med. Devices 11(2), (2014)


Pig (20)

Pig (3)

Dog (24)

Pig (11)

Rat (33)

Rabbit (10)

Dog (12)

Dog (15)

Pig (10)

Dog (12)

Dog (22)

Rat (67)

Liang et al. (2009)

Liang et al. (2010)

Shinar et al. (1998)

Komuro et al. (2002)

Griskchiet et al. (2003)

Jansen et al. (2004)

Nakase et al. (2008)


Kajitani et al. (2001)

Isch et al. (2001)


Lopes et al. (2006)

SIS (pig)

UBM (pig) (peracetic acid)

Alloderm

Aorta (pig) (Triton X-100)

SIS vs UBS (peracetic acid)

PGA-muscularis layer-amniotic membrane

Pvdf Polyglactin 910

Collagen PGA

Collagen PGA

Collagen Polyglactin 910

Nitinol Silicon

Nitinol Collagen

Substitute

0 2

1.5

Cervical Abdominal Patch (½ circumferential)


Cervical Patch (10 20)

Thorax Patch (20)

7

5

No

3

4

Muscularis externa (autologous)

No

No

0 4

12 0

1 0

1 0

4 0

15

No

Cervical Patch (30 50) Circumferential (50)

8 0

15

Fibroblasts/keratinocytes Oral mucosa/autologous Smooth muscle cells/ stomach/autologous


1 3

3

No

Patch Organ units derived esophagus (esophagus rats/heterologous)

3 0

2

No

0 2

6

No

1 0

6 9

Morbidity Mortality

42

6

Follow-up (months)

No

No

Cells (source)

Abdominal Patch (10 5)


Cervical/thorax Circumferential myomectomy (20–30)

Cervical Circumferential (60) Patch (30 25)





Mucosa Submucosa (with conserved muscularis)

Mucosa

Mucosa Muscularis externa





Muscularis


Mucosa Submucosa

Mucosa Submucosa



Animals (n)

Study (year)

Table 5. In vivo and in situ implantation studies of esophageal replacement (cont.).


[87]

[86]

[74]

[90]

[91]

[65]

[70]

[64]

[68]

[69]

[56]

[55]

Ref.


Review

235

236


[99]

Mucosa Submucosa Muscularis externa 0 0 3 BMSCs (autologous BMSCs of dogs) Cervical ½ circumferential (50) Dog (12) Tan et al. (2013)

SIS (Pig) (SDS)

[89]

Mucosa 16 10 12 No Cervical Circumferential (20) Pig (27) Gaujoux et al. (2010)

Aorta (pig)

[88]

0 0 Dog (12) Wei et al. (2009)

SIS (pig) (DEOX)

Cervical ½ circumferential (50)

Epithelial cells (autologous oral mucosal epithelial cells of dogs)

2

Mucosa

[85]

Mucosa 3 3 18 Omentum Abdominal ½ circumferential (40) Rat (27) Urita et al. (2007)

GAM (rat) (DEOX)

0 0 1 Pig (6) Marzaro et al. (2006)

EAM (pig) (DEOX)

Thorax Patch muscularis (20)

Smooth muscle cells (autologous esophagus cells of pigs)

Morbidity Mortality Animals (n)

Substitute


Cells (source)

Follow-up (months)

Muscularis externa

[84]


Study (year)

Table 5. In vivo and in situ implantation studies of esophageal replacement (cont.).



Ref.

Review

abdominal esophagus in rabbits [70]. Nakase et al. studied PGAcoated collagen and described eight complications (stricture) in 12 dogs; the histologic examination of the substitute revealed only mucosal regeneration [65]. Natural scaffolds have been used in esophageal tissue engineering since 2000. Badylak et al. studied SIS versus UBS scaffolds lacking seeded cells in dog models [91]. Four strictures occurred when the tube configuration was implanted. Histological examination of the patch configuration indicated mucosal and muscularis regeneration [91]. This natural scaffold did not resolve the stricture complications. Strictures systematically occurred in all of the studies in which the esophagus was removed circumferentially and replaced by a natural scaffold [86,89,91]. Badylak et al. found that the stricture occurred due to the near lack of intraluminal pressure in the esophagus [86]. Atala et al. demonstrated that a coculture of urothelial cells and smooth muscle cells facilitated tissue growth and inhibited the contraction of the remodeling tissue, which has implications for the formation of strictures [97,98]. Cell seeding could increase the stricture rate [53,54,64,84,88,99], but different types of cells were used in the relevant studies and therefore no definitive conclusions can be drawn (epithelial cells, smooth muscle cells, organ units or stem cells). A recent study of a natural decellularized scaffold seeded with stem cells offered new clinical perspectives [99]. Tan et al. decellularized pig SIS using sodium dodecyl sulfate, seeded the scaffold with bone marrow stem cells, and, subsequently, implanted it in 12 dogs [99]. They obtained re-epithelialization, revascularization and muscular regeneration. Seeding bone marrow stem cells increased the vascular density and squamous epithelial coverage and decreased the inflammatory response. This study [99] showed the potential of stem cells in esophageal tissue engineering. Articles point out natural chemoattractive mechanisms that can bring stem cells from far and near to sites of tissue damage, and capacity to establish regenerative microenvironments [100]. The mesenchymal stem cells had plasticity and can be differentiate of mature phenotypes into wholly different cell types [101]. These advantages are very interesting for substitute different tissues with the same type of cells. There are different methods that have been employed for using stem cells in scaffolds. Stem cells have been seeded into the scaffolds in vitro and, after a short incubation to insure attachment, the cell-scaffold composites were implanted [102]. Another modes consists on the cell-scaffold composite was incubated in differentiation medium to stimulate stem cells progression into a specific lineage; after maturation delay, the composite was implanted [102]. The last approach is to implant scaffolds to which stem cells are able to attach to docking sites or to implant scaffolds with the included cells in protective coats and allow the scaffold to mature in vivo [103]. All of these techniques have resulted in well-integrated, newly differentiated tissue. Although these approaches have been described in various animal models and are limited numbers in esophageal tissue engineering [99]. Industrial production of DM is yet performed by several firms (CookÒ, CovidienÒ, LifecellÒ). Porcine skin is the most Expert Rev. Med. Devices 11(2), (2014)


Review


Table 6. Advantages and inconveniences of biomaterials used in esophageal tissue engineering. Scaffolds

Advantages

Inconveniences

Non-absorbable scaffold

Abundant material (collagen silicone) No product degradation No cytotoxicity Controlled product Reproducible product

Need of stent Stenosis after stent removing No tissue regeneration in large defect

Polymeric scaffolds

Abundant biomaterial Reproducible manufactured Biocompatibility improved in different tissues Resorbable biomaterials Choice of polymers Possibility to control microarchitecture

Necessity to cover polymer with coating of collagen No control of degradation No similar to the native tissue Toxicity of products degradation Scaffold architecture depending of the nature of fabrication Variable types of fabrication

Decellularized matrices

Mimic of the native tissue Respect of architectural environment Mechanical properties similar to the native tissue No immunogenicity Possibility of cells seeding with stem cells or differentiated cells Industrial process

Possibility of cytotoxicity (chemical decellularization) No standardized evaluation of immunogenicity Small number of esophagus decellularized matrix for human used Heterologous matrix Need of cells seeding

common matrix employed in clinical practice (general surgery). SIS is also developed for clinical practice like abdominal wall surgery, colonic surgery or urology [104–106]. In esophageal tissue engineering, specific products are not yet developed for many reasons. First in clinical practice the length of defect is superior to 15 cm (circumferential) and preclinical results are not sufficient for these characteristics. Second, esophageal tissue engineering is not achieved and it is necessary to continue experimental development. Finally, the economic issue for pharmaceutics firm in esophageal tissue engineering has not advantages for the moment. TABLE 5 summarizes the preclinical studies of esophageal tissue engineering and their main results. Expert commentary

In recent decades, despite advances in cancer care, malignancy (squamous cell carcinoma and adenocarcinoma) of the esophagus has maintained a poor prognosis, with 5-year survival rates of 3–40%. Surgical resection remains the cornerstone of any potential cure, but esophagectomy is still associated with high morbidity and mortality despite recent advances in therapeutic modalities. Early postoperative complication rates are in the range of 40–80%, depending on the applied criteria and depending on the American Society of Anesthesiology (ASA) score, age, gender, anastomosis localization and extent of resection. Mortality is reduced in ‘high-volume centers’ due to better perioperative management. However, morbidity is still high and quality of life is low in the surviving patients. Organ transplantation is not helpful in esophageal disorders due to vascularization problems. Thus, tissue engineering would offer many advantages for patients and clinical practitioners. The recent development of decellularized esophageal scaffolds seeded with stem cells may offer new hope. However, the informahealthcare.com

problem of anastomotic stenosis remains when the replacement is circumferential. Healing of the substitute results in scar tissue and is responsible for stenosis. Adding stem cells to the scaffold can limit this process. However, the best types of cells and their specifications have not been clearly defined. Stents can be used for decrease the rate of stenosis. Mechanical stimulation of the internal lumen by the stent might have disturbed epithelial regeneration, and granulation tissue was able to develop only outside the tube. After tube removal, the granulation tissue began to regress, while regeneration of the epithelium was progressing and stenosis in the middle portion of the regenerated esophagus occurred. This phenomenon can be limited depending of the time of stent removed (6 weeks). Stenosis occurred in replacement of tubular organ (not only in esophagus). For example, stenosis occurred in ureteral segment replacement using a circumferential small-intestinal submucosa graft [107]. We think that the development of stenosis occurred when circumferential replacement is performed regardless native tissue and stents cannot apply in clinical practice. Many preclinical studies have used the canine model and the cervical esophagus for anatomical reasons (easy approach and follow-up), but this model is not actually relevant for humans. For our team, the best model is the pig because the length of esophagus, histological characteristics and mechanical properties are similar with human esophagus. The increased rate of carcinoma in the lower esophagus (adenocarcinoma) in humans results in an increased rate of lower esophagectomy. In addition, esophagectomy for carcinoma of the upper esophagus has decreased because it can be cured by radiochemotherapy without surgery. This part of the esophagus has different histological and mechanical properties than the lower esophagus. The major histological difference is in the muscularis externa. In the lower esophagus, the muscularis externa is composed of smooth 237


Review


muscle, whereas it is composed of striated muscle in the upper esophagus. Moreover, the upper esophagus is stronger than the lower esophagus, which may decrease the risk of anastomotic complications in the upper esophagus. Moreover, the greatest challenge is to obtain a functional esophagus. The tissue-engineered esophageal substitutes do not perform peristaltic movement. Innervation of DM was studied [111] in abdominal wall repair and esophageal replacement. Innervation started when muscle tissue was present. In abdominal wall, mature nerves were found within the remodeled DM at 28 and 91 days in the esophageal canine repair [111]. The temporal and spatial association between nerve and muscle suggests that either cell types or their precursor respond to the same stimulus, participate in paracrine interaction and direct cell-to-cell communication [112]. This process is not controlled and the types of nerve cannot distinguish such as motor, sensory or nociceptive. Complete and reproducible innervation of the substitutes remains the challenge of the next few years. TABLE 6 summarizes the advantages and inconveniences of different types of scaffolds. Five-year view

In the future, replacing the esophagus with an artificial substitute will most likely be a therapeutic option for managing

esophageal diseases requiring esophagectomy. New therapies should be superior to current conventional approaches, mainly due to the reduction of short- and long-term morbidity. Progress in esophageal tissue engineering can be achieved only by interactions among biologists, engineers and surgeons. Although decellularization appears to be the best method of preparing a scaffold because it preserves the natural ECM, the following challenges remain: this method does not prevent stenosis after a circumferential replacement; cell seeding of the scaffold may limit the formation of scar tissue; the type of cells that could be seeded is not clear because various cells constitute the esophagus, including epithelial cells, muscular cells and nerve cells; and the differentiated cells or stem cells that would provide the best results remain to be demonstrated. Producing a functional autologous tissue system, such as an implantable autologous esophagus, will require at least 10–15 years of research. Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending or royalties. No writing assistance was utilized in the production of this manuscript.

Key issues • Esophageal tissue engineering has been utilized for more than a century. • Recent developments in scaffold preparation are promising. • Decellularized scaffolds may be superior to manufactured scaffolds. However, they do not decrease long-term morbidity. • The main complication is stenosis of the esophageal substitute, which occurs when the replacement is circumferential. • Supplementation with cells may decrease the stenosis phenomenon. However, the type(s) of cells required has not been clearly defined. • Esophageal substitution in animal models is not similar to that of humans.

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Cell sheets engineering for esophageal regenerative medicine.

Stereolithography in tissue engineering.

Tissue engineering in dentistry.

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Tissue Engineering in Orthopaedics.

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Airway tissue engineering: an update.

3-D biotech: tissue engineering.

Synthetic biology meets tissue engineering.

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