Activin A affects feeding by promoting the inner diameter and muscle development of the pharynx and oesophagus in zebrafish (Danio rerio) larvae.

Activin A belongs to the superfamily of transforming growth factor-ß and plays an important role in hormone regulation and tissue development. However, few research studies have been conducted on the effect of activin A on feeding organs in fish. In this study, the zebrafish (Danio rerio) larvae were treated with 1 ng ml-1 activin A for 8 days continuously. The haematoxylin and eosin (H&E) staining section results revealed that the transverse inner diameter of the pharynx and oesophagus significantly increased on the third and eighth days after treatment compared with the control group (P < 0.05). On the eighth day, the cross-sectional area of the pharyngeal muscle increased by 8638 µm2 compared to the control group (P < 0.05). The RNA in situ hybridization results also showed that the expression of skeletal muscle-specific genes (myog and myod) was significantly increased in pharyngeal muscle on the eighth day. Furthermore, the qRT-PCR results showed the expression of gh gene was significantly increased on the eighth day (P < 0.05). At the same time, more larvae in activin A group were able to feed larger brine shrimp (Artemia) than in the control group on the eighth day. In conclusion, activin A could affect feeding by promoting the inner diameter and muscle development of the pharynx and oesophagus in zebrafish larvae. This study is the first to report that the development of the pharynx and oesophagus can directly affect food intake in fish larvae, which provides a theoretical basis for the study of food intake of fish at an early stage.

A single-progenitor model as the unifying paradigm of epidermal and esophageal epithelial maintenance in mice.

In adult skin epidermis and the epithelium lining the esophagus cells are constantly shed from the tissue surface and replaced by cell division. Tracking genetically labelled cells in transgenic mice has given insight into cell behavior, but conflicting models appear consistent with the results. Here, we use an additional transgenic assay to follow cell division in mouse esophagus and the epidermis at multiple body sites. We find that proliferating cells divide at a similar rate, and place bounds on the distribution cell cycle times. By including these results in a common analytic approach, we show that data from eight lineage tracing experiments is consistent with tissue maintenance by a single population of proliferating cells. The outcome of a given cell division is unpredictable but, on average, the likelihood of producing proliferating and differentiating cells is equal, ensuring cellular homeostasis. These findings are key to understanding squamous epithelial homeostasis and carcinogenesis.

Recent advances in deriving human endodermal tissues from pluripotent stem cells.

The utilization of directed differentiation of human pluripotent stem cells to generate human tissues is quickly evolving. Here we review recent advances in the derivation and applications of human endodermal tissues, including the esophagus, lung, pancreas, liver, stomach, small intestine, and colon. Improvements in tissue transcriptional and functional maturation, multicellular complexity, and scalability allow better development and disease modeling, large-scale drug and toxicity screening, and potentially cell therapeutic applications.

Tissue-Engineered Esophagus via Bioreactor Cultivation for Circumferential Esophageal Reconstruction.

The use of biomaterials for circumferential esophageal repair is technically challenging in a rat model, and an optimal scaffold implantation technique with nutritional support is essential. The purpose of this study was to investigate the effects of three-dimensional printed esophageal grafts and bioreactor cultivation on muscle regeneration and reepithelialization from circumferential esophageal defects in a rat model. Here, we designed an artificial esophagus that can enhance the regeneration of esophageal mucosa and muscle through the optimal combination of a two-layered tubular scaffold and mesenchymal stem cell-based bioreactor system. The graft was verified by the performance comparison with an omentum-cultured esophageal scaffold. We also applied a new surgical anastomosis technique and a thyroid gland flap over the implanted scaffold to improve graft survival. Although no regenerated mucosal layer was observed around the implants of the control group, histological examination of the regenerative esophagi along the scaffold revealed that the bioreactor system and omentum-cultured groups showed more than 80% of the mucosal regeneration without a fistula. The regenerated tissues showed that the integration of the esophageal scaffold and its native esophageal tissue was intact and were covered with layers of stratified squamous epithelium with several newly developed blood vessels. Therefore, this study describes a novel approach for circumferential esophageal reconstruction. Impact Statement In vivo functional esophageal reconstruction remains challenging due to anastomosis site leakage and necrosis of the implanted scaffold in a circumferential esophageal defect. Therefore, it is necessary to develop a tissue-engineered esophagus that enables regeneration of esophageal mucosa and muscle without leakage of the esophageal anastomosis. In this study, we proposed an intriguing strategy that combines a mesenchymal stem cell-seeded tubular scaffold with a bioreactor system for esophageal reconstruction and introduced a new surgical anastomosis technique with the thyroid gland flap over the implanted scaffold to improve graft survival. We believe that this system should be a powerful platform for circumferential replacement of the esophagus in a rat model.

Synchronized mesenchymal cell polarization and differentiation shape the formation of the murine trachea and esophagus.

Tube morphogenesis is essential for internal-organ development, yet the mechanisms regulating tube shape remain unknown. Here, we show that different mechanisms regulate the length and diameter of the murine trachea. First, we found that trachea development progresses via sequential elongation and expansion processes. This starts with a synchronized radial polarization of smooth muscle (SM) progenitor cells with inward Golgi-apparatus displacement regulates tube elongation, controlled by mesenchymal Wnt5a-Ror2 signaling. This radial polarization directs SM progenitor cell migration toward the epithelium, and the resulting subepithelial morphogenesis supports tube elongation to the anteroposterior axis. This radial polarization also regulates esophageal elongation. Subsequently, cartilage development helps expand the tube diameter, which drives epithelial-cell reshaping to determine the optimal lumen shape for efficient respiration. These findings suggest a strategy in which straight-organ tubulogenesis is driven by subepithelial cell polarization and ring cartilage development.

Changes in esophageal mucosal morphology and in growth of breast feeding rats fed with milk of low or high temperature.

Dear Editor, This rather original investigation was performed some time ago but is still unpublished. Its aim was to estimate the effect of low or high temperature cow's milk given to breast feeding rats in order to study possible influence of this diet to rats, to their physical growth and also esophageal mucosal pathology. Ten white female Wistar rats, 20 days of age were separated from their mothers and divided in 2 groups of 5 members each. Five of them were fed with milk kept at 42oC by using a special warming device. The other 5 animals were fed with cold milk kept at 4oC during feeding. The duration of feeding was 34 days. Animals were finally sacrificed with a lethal dose of ether. The two groups were examined and compared. The group of rats fed by the warm milk was better grown as indicated by gaining more body weight, being more active and drinking more milk. Specimens were taken from the middle esophagus and after specific treatment were examined under the electronic microscope. We found: In both groups the most impressive finding in esophageal mucosa was an edematous intercellular space in all epithelial layers with many microorganisms in these layers. Acantholysis was often identified while in other areas a keratin transformation was noticed even in the basic layers, while basic membrane was absent. Epithelial cells showed edematous mitochondria and formation of myelin bodies. Degenerative changes and interstitial edema were noticed in the chorio. The above findings suggest that hot milk but not cold milk improves the growth of the rats studied. Cold and also hot milk had a damaging effect on the rats' esophageal mucosa. It is obvious that many options for further research arise related to the range of temperature of food intake that will not cause damage to gastric epithelium.

Morphology and ultrastructure of the esophagus during the ontogeny of the spider crab Maja brachydactyla (Decapoda, Brachyura, Majidae).

The esophagus of the eucrustaceans is known as a short tube that connects the mouth with the stomach but has generally received little attention by the carcinologists, especially during the larval stages. By this reason, the present study is focused on the morphology and ultrastructure of the esophagus in the brachyuran Maja brachydactyla during the larval development and adult stage. The esophagus shows internally four longitudinal folds. The simple columnar epithelium is covered by a thick cuticle. The epithelial cells of the adults are intensively interdigitated and show abundant apical mitochondria and bundles of filamentous structures. The cuticle surface has microspines and mutually exclusive pores. Three muscle layers surrounded by the connective tissue are reported: circular muscles forming a broad continuous band, longitudinal muscle bundles adjacent to the circular muscles, and dilator muscles crossing the connective tissue vertically toward the epithelium. The connective tissue has rosette glands. The esophagus of the larvae have epithelial cells with big vesicles but poorly developed interdigitations and filamentous structures, the cuticle is formed by a procuticle without differentiated exocuticle and endocuticle, the connective layer is thin and the rosette glands are absent. The observed features can be explained by his role in the swallowing of the food.

Design of a Bioabsorbable Multilayered Patch for Esophagus Tissue Engineering.

A gold standard for esophagus reconstruction is not still available. The present work aims to design a polymer patch combining synthetic polylactide-co-polycaprolacton and chitosan biopolymers, tailoring patch properties to esophageal tissue characteristics by a temperature-induced precipitation method, to get multilayered patches (1L, 2L, and 3L). Characterization shows stable multilayered patches (1L and 2L) by selection of copolymer type, and their M w . In vitro investigation of the functional patch properties in simulated physiologic and pathologic conditions demonstrates that the chitosan layer (patch 3L) decreases patch stability and cell adhesion, while improves cell proliferation. Patches 2L and 3L comply with physiological esophageal pressure (3-5 kPa) and elongation (20%).

Development and stem cells of the esophagus.

The esophagus is derived from the anterior portion of the developmental intermediate foregut, a structure that also gives rise to other organs including the trachea, lung, and stomach. Genetic studies have shown that multiple signaling pathways (e.g. Bmp) and transcription factors (e.g. SOX2) are required for the separation of the esophagus from the neighboring respiratory system. Notably, some of these signaling pathways and transcription factors continue to play essential roles in the subsequent morphogenesis of the esophageal epithelium which undergoes a simple columnar-to-stratified squamous conversion. Reactivation of the relevant signaling pathways has also been associated with pathogenesis of esophageal diseases that affect the epithelium and its stem cells in adults. In this review we will summarize these findings. We will also discuss new data regarding the cell-of-origin for the striated and smooth muscles surrounding the esophagus and how they are differentiated from the mesenchyme during development.

Surface and interfacial creases in a bilayer tubular soft tissue.

Surface and interfacial creases induced by biological growth are common types of instability in soft biological tissues. This study focuses on the criteria for the onset of surface and interfacial creases as well as their morphological evolution in a growing bilayer soft tube within a confined environment. Critical growth ratios for triggering surface and interfacial creases are investigated both analytically and numerically. Analytical interpretations provide preliminary insights into critical stretches and growth ratios for the onset of instability and formation of both surface and interfacial creases. However, the analytical approach cannot predict the evolution pattern of the model after instability; therefore nonlinear finite element simulations are carried out to replicate the poststability morphological patterns of the structure. Analytical and computational simulation results demonstrate that the initial geometry, growth ratio, and shear modulus ratio of the layers are the most influential factors to control surface and interfacial crease formation in this soft tubular bilayer. The competition between the stretch ratios in the free and interfacial surfaces is one of the key driving factors to determine the location of the first crease initiation. These findings may provide some fundamental understanding in the growth modeling of tubular biological tissues such as esophagi and airways as well as offering useful clues into normal and pathological functions of these tissues.

Embracing change: striated-for-smooth muscle replacement in esophagus development.

The esophagus functions to transport food from the oropharyngeal region to the stomach via waves of peristalsis and transient relaxation of the lower esophageal sphincter. The gastrointestinal tract, including the esophagus, is ensheathed by the muscularis externa (ME). However, while the ME of the gastrointestinal tract distal to the esophagus is exclusively smooth muscle, the esophageal ME of many vertebrate species comprises a variable amount of striated muscle. The esophageal ME is initially composed only of smooth muscle, but its developmental maturation involves proximal-to-distal replacement of smooth muscle with striated muscle. This fascinating phenomenon raises two important questions: what is the developmental origin of the striated muscle precursor cells, and what are the cellular and morphogenetic mechanisms underlying the process? Studies addressing these questions have provided controversial answers. In this review, we discuss the development of ideas in this area and recent work that has shed light on these issues. A working model has emerged that should permit deeper understanding of the role of ME development and maturation in esophageal disorders and in the functional and evolutionary underpinnings of the variable degree of esophageal striated myogenesis in vertebrate species.

Strain induced esophageal growth in a novel rodent model.

PURPOSE: Longitudinal esophageal strain has been shown to increase esophageal length but the contribution of tissue hyperplasia to this growth is unknown. We used a novel model of esophageal stretch to determine the cellular response to the strain stimulus. METHODS: Male Sprague-Dawley rats underwent transection of the distal esophagus. The distal stump was ligated and stretched over a silicone tube. The proximal esophageal stump was anastomosed to the stomach to restore continuity. After two, four, or seven days, the silicone tube was removed and the esophageal segment was measured and compared to its initial length. Sham animals had only a thin piece of silicone tubing placed. Standardized histologic sections were evaluated for wall thickness. Immunofluorescence with DAPI, Ki-67, and Myogenin antibodies was used to assess nuclear density, proliferation indices, and myoblast differentiation indices. RESULTS: Experimental animals demonstrated a significant increase in esophageal length compared to sham controls at four and seven days with no difference at two days. There was significant lengthening between four and seven days among the experimental animals. There was no change in wall thickness between experimental and sham animals at any time point. Nuclear density was increased at all time points, although this only reached significance at day four. Proliferation indices were significantly increased relative to sham controls at all time points. Esophageal strain induced significantly increased myoblast differentiation. CONCLUSION: In this novel rat model of esophageal strain, lengthening is associated with stable esophageal wall thickness, increased nuclear density, increased cellular proliferation, and increased myogenin expression. These data suggest that true tissue hyperplasia may contribute to the increased length seen after esophageal strain.

Gastroesophageal reflux and congenital gastrointestinal malformations.

Although the outcome of newborns with surgical congenital diseases (e.g., diaphragmatic hernia; esophageal atresia; omphalocele; gastroschisis) has improved rapidly with recent advances in perinatal intensive care and surgery, infant survivors often require intensive treatment after birth, have prolonged hospitalizations, and, after discharge, may have long-term sequelae including gastro-intestinal comorbidities, above all, gastroesophageal reflux (GER). This condition involves the involuntary retrograde passage of gastric contents into the esophagus, with or without regurgitation or vomiting. It is a well-recognized condition, typical of infants, with an incidence of 85%, which usually resolves after physiological maturation of the lower esophageal sphincter and lengthening of the intra-abdominal esophagus, in the first few months after birth. Although the exact cause of abnormal esophageal function in congenital defects is not clearly understood, it has been hypothesized that common (increased intra-abdominal pressure after closure of the abdominal defect) and/or specific (e.g., motility disturbance of the upper gastrointestinal tract, damage of esophageal peristaltic pump) pathological mechanisms may play a role in the etiology of GER in patients with birth defects. Improvement of knowledge could positively impact the long-term prognosis of patients with surgical congenital diseases. The present manuscript provides a literature review focused on pathological and clinical characteristics of GER in patients who have undergone surgical treatment for congenital abdominal malformations.

Esophageal development and epithelial homeostasis.

The esophagus is a relatively simple organ that evolved to transport food and liquids through the thoracic cavity. It is the only part of the gastrointestinal tract that lacks any metabolic, digestive, or absorptive function. The mucosa of the adult esophagus is covered by a multilayered squamous epithelium with a remarkable similarity to the epithelium of the skin despite the fact that these tissues originate from two different germ layers. Here we review the developmental pathways involved in the establishment of the esophagus and the way these pathways regulate gut-airway separation. We summarize current knowledge of the mechanisms that maintain homeostasis in esophageal epithelial renewal in the adult and the molecular mechanism of the development of Barrett's metaplasia, the precursor lesion to esophageal adenocarcinoma. Finally, we examine the ongoing debate on the hierarchy of esophageal epithelial precursor cells and on the presence or absence of a specific esophageal stem cell population. Together the recent insights into esophageal development and homeostasis suggest that the pathways that establish the esophagus during development also play a role in the maintenance of the adult epithelium. We are beginning to understand how reflux of gastric content and the resulting chronic inflammation can transform the squamous esophageal epithelium to columnar intestinal type metaplasia in Barrett's esophagus.

BMP-driven NRF2 activation in esophageal basal cell differentiation and eosinophilic esophagitis.

Tissue homeostasis requires balanced self-renewal and differentiation of stem/progenitor cells, especially in tissues that are constantly replenished like the esophagus. Disruption of this balance is associated with pathological conditions, including eosinophilic esophagitis (EoE), in which basal progenitor cells become hyperplastic upon proinflammatory stimulation. However, how basal cells respond to the inflammatory environment at the molecular level remains undetermined. We previously reported that the bone morphogenetic protein (BMP) signaling pathway is critical for epithelial morphogenesis in the embryonic esophagus. Here, we address how this pathway regulates tissue homeostasis and EoE development in the adult esophagus. BMP signaling was specifically activated in differentiated squamous epithelium, but not in basal progenitor cells, which express the BMP antagonist follistatin. Previous reports indicate that increased BMP activity promotes Barrett's intestinal differentiation; however, in mice, basal progenitor cell-specific expression of constitutively active BMP promoted squamous differentiation. Moreover, BMP activation increased intracellular ROS levels, initiating an NRF2-mediated oxidative response during basal progenitor cell differentiation. In both a mouse EoE model and human biopsies, reduced squamous differentiation was associated with high levels of follistatin and disrupted BMP/NRF2 pathways. We therefore propose a model in which normal squamous differentiation of basal progenitor cells is mediated by BMP-driven NRF2 activation and basal cell hyperplasia is promoted by disruption of BMP signaling in EoE.

Evaluation of decellularization protocols for production of tubular small intestine submucosa scaffolds for use in oesophageal tissue engineering.

Small intestine submucosa (SIS) has emerged as one of a number of naturally derived extracellular matrix (ECM) biomaterials currently in clinical use. In addition to clinical applications, ECM materials form the basis for a variety of approaches within tissue engineering research. In our preliminary work it was found that SIS can be consistently and reliably made into tubular scaffolds which confer certain potential advantages. Given that decellularization protocols for SIS are applied to sheet-form SIS, it was hypothesized that a tubular-form SIS would behave differently to pre-existing protocols. In this work, tubular SIS was produced and decellularized by the conventional peracetic acid-agitation method, peracetic acid under perfusion along with two commonly used detergent-perfusion protocols. The aim of this was to produce a tubular SIS that was both adequately decellularized and possessing the mechanical properties which would make it a suitable scaffold for oesophageal tissue engineering, which was one of the goals of this work. Analysis was carried out via mechanical tensile testing, DNA quantification, scanning electron and light microscopy, and a metabolic assay, which was used to give an indication of the biocompatibility of each decellularization method. Both peracetic acid protocols were shown to be unsuitable methods with the agitation-protocol-produced SIS, which was poorly decellularized, and the perfusion protocol resulted in poor mechanical properties. Both detergent-based protocols produced well-decellularized SIS, with no adverse mechanical effects; however, one protocol emerged, SDS/Triton X-100, which proved superior in both respects. However, this SIS showed reduced metabolic activity, and this cytotoxic effect was attributed to residual reagents. Consequently, the use of SIS produced using the detergent SD as the decellularization agent was deemed to be the most suitable, although the elimination of the DNase enzyme would give further improvement.

Juvenile megaesophagus in PKCα-deficient mice is associated with an increase in the segment of the distal esophagus lined by smooth muscle cells.

Megaesophagus in mice has been associated with several genetic defects. In the present study we expand the range of genes associated with esophageal function and morphology by protein kinase C alpha (PKCα). PKCα-deficient mice showed a six times increased prevalence of megaesophagus at the age of 9-10 weeks compared to wild-type animals. In contrast, in a restricted number of 14-month-old animals of both genotypes a similar prevalence of megaesophagus was found. Megaesophagus was associated with an increased portion of the distal esophagus lined by smooth muscle cells. Achalasia-like degeneration or loss of neuronal cells, inflammation or fibrosis was not present in any of the animals. The results of the study therefore suggest that PKCα expression is associated with a delayed replacement of embryonic smooth muscle by skeletal muscle at the distal esophagus and consecutive megaesophagus in young mice, which, however, is not present at the same prevalence at an advanced age.

Promoting epithelium regeneration for esophageal tissue engineering through basement membrane reconstitution.

Scaffolds mimicking hierarchical features of native extracellular matrices may facilitate cell growth and anatomical tissue regeneration. In our previous study, esophageal basement membrane (BM) was shown to be composed of interwoven fibers with mean diameter of 66 ± 24 nm (range 28-165 nm) and with abundant pores of unequal sizes. The main extracellular matrix (ECM) contents found in porcine esophageal BM were collagen IV, laminin, entactin, and proteoglycans. In this work, biodegradable polycaprolactone (PCL) and silk fibroin (SF) were spun with electrospinning technology, both individually and in combination, to fabricate fibrous scaffolds with diameters between 64 and 200 nm. The surface morphologies of PCL, PCL/SF, and SF scaffolds were observed under scanning electron microscopy. Their mechanical properties were tested and the cytocompatibility was evaluated in vitro via culture of primary epithelial cells (ECs). The SF or PCL/SF scaffold favorably promoted epithelial cell attachment and proliferation comparing with PCL scaffold. However, mitochondrial activity of epithelial cells was greatly promoted when major BM proteins were coated onto the electrospun scaffold to provide an ECM-like structure. Results from in vivo tests revealed that the electrospun scaffolds coated with BM protein possess good biocompatibility and capability to promote epithelium regeneration.

Effects of internal pressure and surface tension on the growth-induced wrinkling of mucosae.

Surface wrinkling of mucosae is crucial for the biological functions of many living tissues. In this paper, we investigate the instability of a cylindrical tube consisting of a mucosal layer and a submucosal layer. Our attention is focused on the effects of internal pressure and surface tension on the critical condition and mode number of surface wrinkling induced by tissue growth. It is found that the internal pressure plays a stabilizing role but basically has no effect on the critical mode number. Surface tension also stabilizes the system and reduces the critical mode number of surface patterns. Besides, the thinner the mucosal layer, the more significant the effect of surface tension. This work may help gain insights into the surface wrinkling and morphological evolution of such tubular organs as airways and esophagi.