Esophageal Regeneration Research Articles

Risk of Esophagitis in Patients with Limited Stage SCLC Treated with QD or BID Chemoradiotherapy Regimens with Contralateral Esophagus Sparing

Recent randomized phase III trials in limited stage (LS) small cell lung carcinoma (SCLC) demonstrate persistent high rates of grade (G) ≥ 3 esophagitis of 16-19% for 45 Gy BID and 66-70 Gy QD radiotherapy (RT). G3 esophagitis dramatically impairs quality of life and is a barrier to treatment intensification. We previously developed a contralateral esophagus sparing technique (CEST) that reduced esophagitis mainly in patients with locally advanced NSCLC treated with chemoRT to 70 Gy/7 weeks. It is not established whether CEST is effective in LS-SCLC patients, especially those receiving BID RT which may limit mucosal esophageal regeneration during treatment. Here, we report our single-institution experience with this approach. We retrospectively analyzed the records of patients with LS-SCLC who received chemoRT between June 2014 and October 2022. Patients were treated with QD or BID schedules at physician discretion though QD has historically been favored at our institution. The esophageal wall contralateral to the tumor was contoured as an avoidance structure. IMRT or VMAT was used to drive steep dose fall-off across the esophagus while maintaining full tumor coverage. Daily cone-beam CT guidance was employed. Toxicity was assessed using CTCAE v5 criteria. Descriptive statistics were used to analyze outcomes. The study was approved by the IRB. We identified 38 consecutive patients with LS-SCLC treated with definitive chemoRT. Median age was 67 years, and 60.5% were female. Twenty-seven were treated with QD RT to a median dose of 63 Gy (range 55.8-70) in 33 fractions (range 31-35). All remaining patients received BID RT 45 Gy in 30 fractions. Median CTV for the QD and BID cohorts was 205 cc and 214 cc, respectively. Almost all patients (n = 37) had tumor located within 1 cm of the esophagus. There were no ≥G3 esophagitis events (95% CI, 0-9.3%). The rate of G2 esophagitis was 21.1% (11.1-36.3%), with no significant difference between QD (26%) and BID (9%) cohorts (p = 0.3). Only two patients required narcotics. For BID patients, CE planning constraints were scaled to reflect the shortened RT course. The median CE maximum dose, V20, and V30 were 44.8 Gy, 7.9 cc, and 6.4 cc, respectively. For the QD cohort, the median CE maximum dose, V45, and V55 were 59.5 Gy, 3.0 cc, and 0.6 cc, respectively. Treatment breaks occurred in nine QD patients due to neutropenia. Median follow-up was 31.0 months. 3-year overall survival was 70.0% (95% CI, 49.8-83.3%). While we are limited by small patient numbers and possible selection bias, our data suggest that CE sparing can be effective in LS-SCLC, with low severe esophagitis risk even for BID RT regimens. CEST is readily adaptable into clinical practice which should facilitate further refinement of empirically derived CE planning constraints.

DNA Methylation Dynamics During Esophageal Epithelial Regeneration Following Repair with Acellular Silk Fibroin Grafts in Rat.

Esophageal pathologies such as atresia and benign strictures often require surgical reconstruction with autologous tissues to restore organ continuity. Complications such as donor site morbidity and limited tissue availability have spurred the development of acellular grafts for esophageal tissue replacement. Acellular biomaterials for esophageal repair rely on the activation of intrinsic regenerative mechanisms to mediate de novo tissue formation at implantation sites. Previous research has identified signaling cascades involved in neoepithelial formation in a rat model of onlay esophagoplasty with acellular silk fibroin grafts, including phosphoinositide 3-kinase (PI3K), and protein kinase B (Akt) signaling. However, it is currently unknown how these mechanisms are governed by DNA methylation (DNAme) during esophageal wound healing processes. Reduced-representation bisulfite sequencingis performed to characterize temporal DNAme dynamics in host and regenerated tissues up to 1 week postimplantation. Overall, global hypermethylation is observed at postreconstruction timepoints and an inverse correlation between promoter DNAme and the expression levels of differentially expressed proteins during regeneration. Site-specific hypomethylation targets genes associated with immune activation, while hypermethylation occurs within gene bodies encoding PI3K-Akt signaling components during the tissue remodeling period. The data provide insight into the epigenetic mechanisms during esophageal regeneration following surgical repair with acellular grafts.

Decellularized esophageal tubular scaffold microperforated by quantum molecular resonance technology and seeded with mesenchymal stromal cells for tissue engineering esophageal regeneration.

Current surgical options for patients requiring esophageal replacement suffer from several limitations and do not assure a satisfactory quality of life. Tissue engineering techniques for the creation of customized “self-developing” esophageal substitutes, which are obtained by seeding autologous cells on artificial or natural scaffolds, allow simplifying surgical procedures and achieving good clinical outcomes. In this context, an appealing approach is based on the exploitation of decellularized tissues as biological matrices to be colonized by the appropriate cell types to regenerate the desired organs. With specific regard to the esophagus, the presence of a thick connective texture in the decellularized scaffold hampers an adequate penetration and spatial distribution of cells. In the present work, the Quantum Molecular Resonance® (QMR) technology was used to create a regular microchannel structure inside the connective tissue of full-thickness decellularized tubular porcine esophagi to facilitate a diffuse and uniform spreading of seeded mesenchymal stromal cells within the scaffold. Esophageal samples were thoroughly characterized before and after decellularization and microperforation in terms of residual DNA content, matrix composition, structure and biomechanical features. The scaffold was seeded with mesenchymal stromal cells under dynamic conditions, to assess the ability to be repopulated before its implantation in a large animal model. At the end of the procedure, they resemble the original esophagus, preserving the characteristic multilayer composition and maintaining biomechanical properties adequate for surgery. After the sacrifice we had histological and immunohistochemical evidence of the full-thickness regeneration of the esophageal wall, resembling the native organ. These results suggest the QMR microperforated decellularized esophageal scaffold as a promising device for esophagus regeneration in patients needing esophageal substitution.

Regeneration of Trachea and Esophagus by Scaffold-Free Structure, Using Bio-3D Printer

Regeneration of Trachea and Esophagus by Scaffold-Free Structure, Using Bio-3D Printer

Chitosan/collagen layer-by-layer deposition for improving the esophageal regeneration ability of nanofibrous mats

The morbidity of a diverse array of esophageal diseases has been increasing in recent years, and postoperative bacterial infection is a common potential risk. Current research on artificial esophageal stents is lacking and less than satisfactory. Artificial materials with excellent mechanical, antimicrobial, and cytocompatible properties at the same time still have great potential. In this study, chitosan (CS) and collagen (COL) were deposited on nylon 6 (N6)/silk fibroin (SF) composite nanofibers using layer-by-layer self-assembly (LBL). The obtained nanofibrous mats were characterized, and the results indicated that the surface morphology and hydrophilicity of the composite nanofibrous mats were obviously improved by the deposition of CS and COL. The mechanical properties of the composite nanofibrous mats were also improved after LBL modification. In addition, the LBL-structured nanofibrous mats possessed excellent antibacterial activity, reaching more than 90% inhibition of gram-positive and negative bacteria. The results of cell experiments indicated that the nanofibrous mats modified by CS and COL had excellent biocompatibility for promoting human esophageal epithelial cell adhesion and proliferation. In vivo animal experiments showed that the LBL-structured nanofibrous mats were beneficial to the healing of esophageal defects compared with the control group. In summary, LBL-structured nanofibrous mats are very promising materials for esophageal replacement to reduce the incidence of postoperative complications.

Esophageal regeneration following surgical implantation of a tissue engineered esophageal implant in a pediatric model

Diseases of the esophagus, damage of the esophagus due to injury or congenital defects during fetal esophageal development, i.e., esophageal atresia (EA), typically require surgical intervention to restore esophageal continuity. The development of tissue engineered tubular structures would improve the treatment options for these conditions by providing an alternative that is organ sparing and can be manufactured to fit the exact dimensions of the defect. An autologous tissue engineered Cellspan Esophageal ImplantTM (CEI) was surgically implanted into piglets that underwent surgical resection of the esophagus. Multiple survival time points, post-implantation, were analyzed histologically to understand the tissue architecture and time course of the regeneration process. In addition, we investigated CT imaging as an “in-life” monitoring protocol to assess tissue regeneration. We also utilized a clinically relevant animal management paradigm that was essential for long term survival. Following implantation, CT imaging revealed early tissue deposition and the formation of a contiguous tissue conduit. Endoscopic evaluation at multiple time points revealed complete epithelialization of the lumenal surface by day 90. Histologic evaluation at several necropsy time points, post-implantation, determined the time course of tissue regeneration and demonstrated that the tissue continues to remodel over the course of a 1-year survival time period, resulting in the development of esophageal structural features, including the mucosal epithelium, muscularis mucosae, lamina propria, as well as smooth muscle proliferation/migration initiating the formation of a laminated adventitia. Long term survival (1 year) demonstrated restoration of oral nutrition, normal animal growth and the overall safety of this treatment regimen.

Three-Dimensional Printed Design of Antibiotic-Releasing Esophageal Patches for Antimicrobial Activity Prevention.

Pharyngoesophageal defects can cause exposure to various bacterial flora and severe inflammation. We fabricated a biodegradable polycaprolactone (PCL) patch composed of both thin film and three-dimensional (3D) printed lattice, and then investigated the efficacy of pharyngoesophageal reconstruction by using 3D printed antibiotic-releasing PCL patches that inhibited early inflammation by sustained tetracycline (TCN) release from both thin PCL films and printed rods implanted in esophageal partial defects. PCL was 3D printed in lattice form on a presolution casted PCL thin film at ∼100 μm resolution. TCN was loaded onto the PCL-printed patches by 3D printing a mixture of TCN and PCL particles melted at 100°C. TCN exhibited sustained release in vitro for over 1 month. After loading TCN, the patches showed decreased tensile strength and Young's modulus, and less than 20% TCN was slowly released from the 2.5% TCN-loaded PCL patches over 150 days. Cytotoxicity tests of extract solutions from patch samples demonstrated excellent in vitro cell compatibility. Antibiotic-releasing PCL patches were then transplanted into partial esophageal defects in rats. Microcomputed tomography analysis revealed no leak of orally injected contrast agent in the entire esophagus. Tissue remodeling was examined through histological responses of M1 and M2 macrophages. In particular, the 1% and 3% TCN patch groups exhibited significant muscle layer regeneration by desmin immunostaining. Further histological and immunofluorescence analyses revealed that the 1% and 3% TCN patch groups exhibited the best esophageal regeneration according to reepithelialization, neovascularization, and elastin texture around the implanted sites. Our antibiotic-releasing patch successfully consolidates the regenerative potential of esophageal muscle and mucosa and the antibacterial activity of TCN for 3D esophageal reconstruction. Impact statement Anastomosis site leakage and necrosis after pharyngoesophageal transplantation inevitably causes mortality because the mediastinum and neck compartments become contaminated. Herein, we present antibiotic-releasing pharyngoesophageal patch that prevents saliva leakage and has an antimicrobial effect. We have demonstrated antibiotic release profile and mechanical properties for esophageal transplantation. Upon esophageal transplantation of antibiotic-releasing polycaprolactone patches, antimicrobial effects and muscle regeneration around the graft sites were clearly identified in the group containing 1% and 3% of tetracycline. The esophageal graft led to the remarkable recovery throughout reepithelialization, neovascularization, and elastin texture of around the implanted sites. We believe that current system is capable of various applications that require antibacterial in vivo.

Molecular mechanisms of esophageal epithelial regeneration following repair of surgical defects with acellular silk fibroin grafts

Constructive remodeling of focal esophageal defects with biodegradable acellular grafts relies on the ability of host progenitor cell populations to repopulate implant regions and facilitate growth of de novo functional tissue. Intrinsic molecular mechanisms governing esophageal repair processes following biomaterial-based, surgical reconstruction is largely unknown. In the present study, we utilized mass spectrometry-based quantitative proteomics and in silico pathway evaluations to identify signaling cascades which were significantly activated during neoepithelial formation in a Sprague Dawley rat model of onlay esophagoplasty with acellular silk fibroin scaffolds. Pharmacologic inhibitor and rescue experiments revealed that epithelialization of neotissues is significantly dependent in part on pro-survival stimuli capable of suppressing caspase activity in epithelial progenitors via activation of hepatocyte growth factor receptor (c-MET), tropomyosin receptor kinase A (TrkA), phosphoinositide 3-kinase (PI3K), and protein kinase B (Akt) signaling mechanisms. These data highlight the molecular machinery involved in esophageal epithelial regeneration following surgical repair with acellular implants.

Regeneration of irradiation-damaged esophagus by local delivery of mesenchymal stem-cell spheroids encapsulated in a hyaluronic-acid-based hydrogel.

Radiation therapy (RT) is a typical treatment for head and neck cancers. Generally, prolonged irradiation of the esophagus causes esophageal fibrosis due to increased reactive oxygen species and proinflammatory cytokines. This study was designed to determine whether catechol-functionalized hyaluronic acid (HA-CA) hydrogel-encapsulated human mesenchymal stem-cell spheroids (MSC-SPs) could ameliorate damage to the esophagus in a mouse model of radiation-induced esophageal fibrosis. MSC-SPs were cultured in concave microwells 600 μm in diameter at a cell density of 1 × 106 cells per mL. Most cells formed spheroids with a 100-300 μm size distribution in concave microwells. MSC-SPs were well maintained in the HA gel, and live-dead staining confirmed that most cells survived. The HA gel containing the MSC-SPs was then injected into the damaged esophageal layer. Inflammatory signs or adverse tissue reactions were not observed after esophageal injection of HA-gel-encapsulated MSC-SPs. Based on Masson's trichrome staining at 4 and 12 weeks postinjection, the inner esophageal layer (IEL) was significantly thinner in the MSC-SP + HA gel group compared to those in the other experimental groups. While the saline and HA gel treatments made the esophageal muscles loose and thick, the MSC-SP + HA gel group showed bundles of tightly packed esophageal muscles, as assayed by desmin immunostaining. qPCR analysis showed that epithelial genes tended to increase over time in the MSC-SP + HA gel group, and the expression of most fibrosis-related genes decreased. This study proposes the potential of using HA-CA-hydrogel-encapsulated MSC-SPs as a promising therapy against radiation-induced esophageal fibrosis.

Multi-layered Free-form 3D Cell-printed Tubular Construct with Decellularized Inner and Outer Esophageal Tissue-derived Bioinks

The incidences of various esophageal diseases (e.g., congenital esophageal stenosis, tracheoesophageal fistula, esophageal atresia, esophageal cancer) are increasing, but esophageal tissue is difficult to be recovered because of its weak regenerative capability. There are no commercialized off-the-shelf alternatives to current esophageal reconstruction and regeneration methods. Surgeons usually use ectopic conduit tissues including stomach and intestine, presumably inducing donor site morbidity and severe complications. To date, polymer-based esophageal substitutes have been studied as an alternative. However, the fabrication techniques are nearly limited to creating only cylindrical outer shapes with the help of additional apparatus (e.g., mandrels for electrospinning) and are unable to recapitulate multi-layered characteristic or complex-shaped inner architectures. 3D bioprinting is known as a suitable method to fabricate complex free-form tubular structures with desired pore characteristic. In this study, we developed a extrusion-based 3D printing technique to control the size and the shape of the pore in a single extrusion process, so that the fabricated structure has a higher flexibility than that fabricated in the conventional process. Based on this suggested technique, we developed a bioprinted 3D esophageal structure with multi-layered features and converged with biochemical microenvironmental cues of esophageal tissue by using decellularizedbioinks from mucosal and muscular layers of native esophageal tissues. The two types of esophageal tissue derived-decellularized extracellular matrix bioinks can mimic the inherent components and composition of original tissues with layer specificity. This structure can be applied to full-thickness circumferential esophageal defects and esophageal regeneration.

Biological properties of a bionic scaffold for esophageal tissue engineering research

Polyurethane is a good matrix material with wide application prospects in tissue engineering because of its adjustable and mechanical properties. A novel biodegradable crosslinked poly(ester urethane) (CPU) with flexible poly(caprolactone) (PCL) and hydrophilic poly(ethylene glycol) (PEG) components has been synthesized using a ferric iron catalyst in our laboratory. In the present study, to promote the interaction between the CPU material and cells, the material was superficially modified by silk fibroin (SF) grafting using an aminolysis and glutaraldehyde crosslinking method to achieve a biocompatible material, CPU-SF. Considering the esophageal-specific architecture, three types of scaffolds were fabricated. S1 was a CPU-SF channel (200 μm in diameter and 30 μm in depth with 30 μm of wall thickness) to support muscle regeneration; S2 was the decellularized matrix of the esophageal mucosa/submucosa obtained by enzyme treatment; and S3 was a combination of S1 and S2, aiming to promote esophageal regeneration with histological structure and function. The biological properties and functions of the materials and scaffolds were investigated by qualitative and quantitative analyses using scanning electron microscopy, immunofluorescence staining, cell adhesion and proliferation measurements, and western blotting technology. The results showed that esophageal smooth muscle cells (SMCs) and epithelial cells (ECs) were very well supported by the scaffolds. In particular, SMCs exhibited guided directional growth and ECs infiltrated the acellular mucosa with retained biological functions when co-cultured on the composite scaffold S3. These findings suggest that the composite bionic scaffold will be a good alternative for esophageal replacement.

Nondestructive Measurement of Regenerated Esophageal Biaxial Mechanical Properties Utilizing Sonometry For Determination of Directional Esophageal Regeneration

Esophageal diseases often necessitate resection of tissue. Current reconstruction options using autologous conduits such as stomach, colon, and small bowel have significant morbidity. Given these limitations in current therapy, we have developed a large animal model to regenerate short segments of esophageal tissue after circumferential full thickness resection. This is performed by implantation of an electrospun polyurethane tissue engineered graft which results in esophageal regrowth. These implantations have been performed both with cell based seeding the polymer scaffold and without with comparable regenerative results. It is possible that esophageal regeneration in this model has unequal contributions from either the proximal or distal esophagus. To determine preferential directionality of regeneration we aim to utilize biomechanical markers to suggest that there are unequal proximal and distal contributions to regenerated tissue. Given limited availability of regenerated tissue, use of a non‐destructive method to perform this testing would have significant utility. Through using our previously validated non‐destructive sonometric ex vivo method we aim to determine differences in mechanical behavior of the regenerated tissue in comparison to proximal and distal esophageal tissues. Using this method we are able to determine longitudinal and circumferential moduli and derive strain energy functions and parameters. Within this work we demonstrate that there are differences in mechanical behavior and that the regenerated tissue behaves more comparably to proximal esophageal tissue suggesting preferential regrowth and unequal contribution to regenerated tissue.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Regeneration of esophagus using a scaffold-free biomimetic structure created with bio-three-dimensional printing.

Various strategies have been attempted to replace esophageal defects with natural or artificial substitutes using tissue engineering. However, these methods have not yet reached clinical application because of the high risks related to their immunogenicity or insufficient biocompatibility. In this study, we developed a scaffold-free structure with a mixture of cell types using bio-three-dimensional (3D) printing technology and assessed its characteristics in vitro and in vivo after transplantation into rats. Normal human dermal fibroblasts, human esophageal smooth muscle cells, human bone marrow-derived mesenchymal stem cells, and human umbilical vein endothelial cells were purchased and used as a cell source. After the preparation of multicellular spheroids, esophageal-like tube structures were prepared by bio-3D printing. The structures were matured in a bioreactor and transplanted into 10-12-week-old F344 male rats as esophageal grafts under general anesthesia. Mechanical and histochemical assessment of the structures were performed. Among 4 types of structures evaluated, those with the larger proportion of mesenchymal stem cells tended to show greater strength and expansion on mechanical testing and highly expressed α-smooth muscle actin and vascular endothelial growth factor on immunohistochemistry. Therefore, the structure with the larger proportion of mesenchymal stem cells was selected for transplantation. The scaffold-free structures had sufficient strength for transplantation between the esophagus and stomach using silicon stents. The structures were maintained in vivo for 30 days after transplantation. Smooth muscle cells were maintained, and flat epithelium extended and covered the inner surface of the lumen. Food had also passed through the structure. These results suggested that the esophagus-like scaffold-free tubular structures created using bio-3D printing could hold promise as a substitute for the repair of esophageal defects.

Polyurethane scaffolds seeded with autologous cells can regenerate long esophageal gaps: An esophageal atresia treatment model

BackgroundPediatric patients suffering from long gap esophageal defects or injuries are in desperate need of innovative treatment options. Our study demonstrates that two different cell sources can adhere to and proliferate on a retrievable synthetic scaffold. In feasibility testing of translational applicability, these cell seeded scaffolds were implanted into piglets and demonstrated esophageal regeneration. MethodsEither porcine esophageal epithelial cells or porcine amniotic fluid was obtained and cultured in 3 dimensions on a polyurethane scaffold (Biostage). The amniotic fluid was obtained prior to birth of the piglet and was a source of mesenchymal stem cells (AF-MSC). Scaffolds that had been seeded were implanted into their respective Yucatan mini-swine. The cell seeded scaffolds in the bioreactor were evaluated for cell viability, proliferation, genotypic expression, and metabolism. Feasibility studies with implantation evaluated tissue regeneration and functional recovery of the esophagus. ResultsBoth cell types seeded onto scaffolds in the bioreactor demonstrated viability, adherence and metabolism over time. The seeded scaffolds demonstrated increased expression of VEGF after 6 days in culture. Once implanted, endoscopy 3 weeks after surgery revealed an extruded scaffold with newly regenerated tissue. Both cell seeded scaffolds demonstrated epithelial and muscle regeneration and the piglets were able to eat and grow over time. ConclusionsAutologous esophageal epithelial cells or maternal AF-MSC can be cultured on a 3D scaffold in a bioreactor. These cells maintain viability, proliferation, and adherence over time. Implantation into piglets demonstrated esophageal regeneration with extrusion of the scaffold. This sets the stage for translational application in a neonatal model of esophageal atresia.

Long-term regeneration and remodeling of the pig esophagus after circumferential resection using a retrievable synthetic scaffold carrying autologous cells

Treatment of esophageal disease can necessitate resection and reconstruction of the esophagus. Current reconstruction approaches are limited to utilization of an autologous conduit such as stomach, small bowel, or colon. A tissue engineered construct providing an alternative for esophageal replacement in circumferential, full thickness resection would have significant clinical applications. In the current study, we demonstrate that regeneration of esophageal tissue is feasible and reproducible in a large animal model using synthetic polyurethane electro-spun grafts seeded with autologous adipose-derived mesenchymal stem cells (aMSCs) and a disposable bioreactor. The scaffolds were not incorporated into the regrown esophageal tissue and were retrieved endoscopically. Animals underwent adipose tissue biopsy to harvest and expand autologous aMSCs for seeding on electro-spun polyurethane conduits in a bioreactor. Anesthetized pigs underwent full thickness circumferential resection of the mid-lower thoracic esophagus followed by implantation of the cell seeded scaffold. Results from these animals showed gradual structural regrowth of endogenous esophageal tissue, including squamous esophageal mucosa, submucosa, and smooth muscle layers with blood vessel formation. Scaffolds carrying autologous adipose-derived mesenchymal stem cells may provide an alternative to the use of a gastro-intestinal conduit for some patients following resection of the esophagus.

Circumferential Esophageal Replacement by a Tissue-engineered Substitute Using Mesenchymal Stem Cells

Tissue engineering appears promising as an alternative technique for esophageal replacement. Mesenchymal stem cells (MSCs) could be of interest for esophageal regeneration. Evaluation of the ability of an acellular matrix seeded with autologous MSCs to promote tissue remodeling toward an esophageal phenotype after circumferential replacement of the esophagus in a mini pig model. A 3 cm long circumferential replacement of the abdominal esophagus was performed with an MSC-seeded matrix (MSC group, n = 10) versus a matrix alone (control group, n = 10), which has previously been matured into the great omentum. The graft area was covered with an esophageal removable stent. A comparative histological analysis of the graft area after animals were euthanized sequentially is the primary outcome of the study. Histological findings after maturation, overall animal survival, and postoperative morbidity were also compared between groups. At postoperative day 45 (POD 45), a mature squamous epithelium covering the entire surface of the graft area was observed in all the MSC group specimens but in none of the control group before POD 95. Starting at POD 45, desmin positive cells were seen in the graft area in the MSC group but never in the control group. There were no differences between groups in the incidence of surgical complications and postoperative death. In this model, MSCs accelerate the mature re-epitheliazation and early initiation of muscle cell colonization. Further studies will focus on the use of cell tracking tools in order to analyze the becoming of these cells and the mechanisms involved in this tissue regeneration.

Full-thickness oesophageal regeneration in pig using a polyurethane mucosal cell seeded graft

Malignant oesophageal pathology typically requires resection of a portion of oesophagus. The aim of this study was to investigate attachment and growth of swine oesophageal mucosal cells on electrospun synthetic nanofibre matrices of varying chemistries and to determine whether a mucosal-seeded graft, in a swine animal model, could induce regeneration. Swine mucosal oesophageal cells were isolated and seeded them onto five different matrix materials. Matrix samples were cultured for up to 14days, after which matrices were analysed for cell attachment. Attachment varied for each of the matrix materials tested, with the most rigid showing the lowest levels of attachment. Importantly, sections of these matrices illustrated that multiple layers of mucosal cells formed, mimicking endogenous oesophageal structure. A tdTomato reporter line (mucosaltdt cells) was created to enable cell tracking. As polyurethane matrix was found optimal through in vitro testing, a graft was prepared using mucosaltdt cells, along with an unseeded control, and implanted into swine for determination of oesophageal regeneration. Mucosal seeded polyurethane grafts initiated full thickness regeneration of the oesophagus, including epithelial, submucosal, and skeletal muscle layers which were highly vascularized. Interestingly, an unseeded graft showed similar regeneration, indicating that the role of cells in the process of oesophageal regeneration is still unclear. The electrospun polyurethane matrix does appear suitable for multilayered cellular attachment and growth of oesophageal mucosal cells, and implantation of polyurethane grafts initiated full thickness regeneration of the oesophagus, indicating potential for oesophageal reconstruction in humans. Copyright © 2016 John Wiley & Sons, Ltd.

Regenerative medicine: Successful in vivo oesophageal regeneration in a human.

Regenerative medicine: Successful in vivo oesophageal regeneration in a human.

Interaction of human smooth muscle cells on random and aligned nanofibrous scaffolds of PHBV and PHBV-gelatin

ABSTRACTTissue-engineered scaffolds with nanofibrous morphology have been shown to be effective in regeneration of tissues because nanofibers mimic the native architecture of the extracellular matrix. The unique alignment in the native tissue motivated the authors to fabricate aligned nanofibers of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and PHBV-gelatin. The in vitro potential of the scaffolds was evaluated using human smooth muscle cells. MTS study confirmed that PHBV aligned nanofibrous scaffold promotes better cell proliferation as well as gene expression of key contractile and extracellular matrix markers than their PHBV-gelatin counterparts. Hence, the PHBV aligned nanofibers can be used as a biomimetic scaffold for the regeneration of esophagus. Electrospinning system for aligned nanofibers fabrication (A) and interaction of human smooth muscle cells on aligned nanofibers (B).

Dry acellular oesophageal matrix prepared by supercritical carbon dioxide

Recently, the use of acellular matrices in tissue engineering has become extremely significant as tissue substitute for organ/tissue reconstruction. In this clinical scenario, banking decellularised organs ready for transplantation would be mandatory for patients with emergency needs.In this work a new process based on supercritical carbon dioxide (SC-CO2) drying technique was investigated for obtaining a dry/preserved decellularized oesophagus. Experiments were performed coupling a detergent enzymatic treatment with two different protocols: (i) SC-CO2 drying; (ii) dehydration in ethanol and a subsequent SC-CO2 drying. The efficiency of the treatments was investigated by monitoring the loss of weight of the treated samples and the maintenance of the extracellular matrix (ECM) architecture, composition and mechanical properties after rehydration.A successful dry acellular matrix was reached in a shorter time using the combined ethanol and SC-CO2 treatment. Histological analysis reported the maintenance of the tissue matrix architecture and the collagen content for all the treated samples, while the preservation of ultrastructural features were confirmed by scanning electron microscopy (SEM). Tensile tests did not show significant differences in terms of fracture strength before and after the supercritical process. Furthermore, the scaffolds demonstrated good biocompatible properties in terms of cell culture viability in vitro. Overall, the results highlighted the potential of this novel technology to obtain a dried acellular matrix for oesophageal regeneration, preserving the extracellular matrix structure of the native tissue.