Decellularized and matured esophageal scaffold for circumferential esophagus replacement: Proof of concept in a pig model (original) (raw)
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Bioengineering
Background: Current esophageal treatment is associated with significant morbidity. The gold standard therapeutic strategies are stomach interposition or autografts derived from the jejunum and colon. However, severe adverse reactions, such as esophageal leakage, stenosis and infection, accompany the above treatments, which, most times, are life threating. The aim of this study was the optimization of a decellularization protocol in order to develop a proper esophageal tissue engineered construct. Methods: Rat esophagi were obtained from animals and were decellularized. The decellularization process involved the use of 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) and sodium dodecyl sulfate (SDS) buffers for 6 h each, followed by incubation in a serum medium. The whole process involved two decellularization cycles. Then, a histological analysis was performed. In addition, the amounts of collagen, sulphated glycosaminoglycans and DNA content were quantified. Resul...
Cells
Esophageal reconstruction through bio-engineered allografts that highly resemble the peculiar properties of the tissue extracellular matrix (ECM) is a prospective strategy to overcome the limitations of current surgical approaches. In this work, human esophagus was decellularized for the first time in the literature by comparing three detergent-enzymatic protocols. After decellularization, residual DNA quantification and histological analyses showed that all protocols efficiently removed cells, DNA (<50 ng/mg of tissue) and muscle fibers, preserving collagen/elastin components. The glycosaminoglycan fraction was maintained (70–98%) in the decellularized versus native tissues, while immunohistochemistry showed unchanged expression of specific ECM markers (collagen IV, laminin). The proteomic signature of acellular esophagi corroborated the retention of structural collagens, basement membrane and matrix–cell interaction proteins. Conversely, decellularization led to the loss of HLA...
Evaluation of decellularized esophagus as a scaffold for cultured esophageal epithelial cells
Journal of Biomedical Materials Research Part A, 2006
Recently, decellularized tissue has been reported to have the potential to regenerate a variety of tissues. However, the optimal protocol for a decellularized esophagus has not been studied. Here, we investigated the effect of different decellularization protocols on the histology and biocompatibility of decellularized esophagi in view of future applications to tissue engineering. The esophageal mucosal epithelium (EP) from 4-week-old Wistar rats was enzymatically dissociated and cultured with growth-arrested feeder cells. Two methods for decellularization using deoxycholic acid (DEOX) or Triton X-100 (TRITON) were compared on esophagi from adult Wistar rats. Those treated with DEOX showed superior mechanical properties, maintenance of extracellular matrix, and lower DNA content than those treated with TRITON. To evaluate the biocompatibility of the scaffold, cultured (passage 3) esophageal epithelial cells were seeded inside the decellularized esophagus and cultured for 7 days. The cells seeded onto the decellularized esophagus were examined histologically and immunocytochemically. Esophageal epithelial cells were stratified into three to four cellular layers in vitro inside the decellularized esophagus, to show polarity. The results from immunocytochemistry indicated that the seeded epithelial cells expressed characteristic marker proteins for native esophageal EP. Decellularized esophagus showed suitable compatibility as a scaffold material for esophageal tissue engineering.
Scientific reports, 2018
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 th...
In Vitro Regeneration of Decellularized Pig Esophagus Using Human Amniotic Stem Cells
BioResearch open access, 2020
Decellularization of esophagus was studied using three different protocols. The sodium deoxycholate/DNase-I (SDC/DNase-I) method was the most successful as evidenced by histology and DNA quantification of the acellular scaffolds. Acellular scaffolds were further analyzed and compared with native tissue by histology, quantitative analysis of DNA, and extracellular matrix (ECM) proteins. Histologically, the SDC/DNase-I protocol effectively produced scaffold with preserved structural architecture similar to native tissue architecture devoid of any cell nucleus. ECM proteins, such as collagen, elastin, and glycosaminoglycans were present even after detergentenzymatic decellularization. Immunohistochemical analysis of acellular scaffold showed weak expression of Gal 1, 3 Gal epitope compared with native tissue. For performing recellularization, human amnion-derived mesenchymal stem cells (MSCs) and epithelial cells were seeded onto acellular esophagus in a perfusion-rotation bioreactor. In recellularized esophagus, immunohistochemistry showed infiltration of MSCs from adventitia into the muscularis externa and differentiation of MSCs into the smooth muscle actin and few endothelial cells (CD31). Our study demonstrates successful preparation and characterization of a decellularized esophagus with reduced load of Gal 1, 3 Gal epitope with preserved architecture and ECM proteins similar to native tissue. Upon subsequent recellularization, xenogeneic acellular esophagus also supported stem cell growth and partial differentiation of stem cells. Hence, the current study offers the hope for preparing a tissue-engineered esophagus in vitro which can be transplanted further into pigs for further in vivo evaluation.
Esophageal tissue engineering: An in-depth review on scaffold design
Biotechnology and Bioengineering, 2012
Treatment of esophageal cancer often requires surgical procedures that involve removal. The current approaches to restore esophageal continuity however, are known to have limitations which may not result in full functional recovery. In theory, using a tissue engineered esophagus developed from the patient's own cells to replace the removed esophageal segment can be the ideal method of reconstruction. One of the key elements involved in the tissue engineering process is the scaffold which acts as a template for organization of cells and tissue development. While a number of scaffolds range from traditional nonbiodegradable tubing to bioactive decellularized matrix have been proposed to engineer the esophagus in the past decade, results are still not yet favorable with many challenges relating to tissue quality need to be met improvements. The success of new esophageal tissue formation will ultimately depend on the success of the scaffold being able to meet the essential requirements specific to the esophageal tissue. Here, the design of the scaffold and its fabrication approaches are reviewed. In this paper, we review the current state of development in bioengineering the esophagus with particular emphasis on scaffold design.
In Vitro Regeneration of Decellularized Pig Esophagus Using Human Amniotic Stem Cells
BioResearch Open Access, 2020
Decellularization of esophagus was studied using three different protocols. The sodium deoxycholate/DNase-I (SDC/DNase-I) method was the most successful as evidenced by histology and DNA quantification of the acellular scaffolds. Acellular scaffolds were further analyzed and compared with native tissue by histology, quantitative analysis of DNA, and extracellular matrix (ECM) proteins. Histologically, the SDC/DNase-I protocol effectively produced scaffold with preserved structural architecture similar to native tissue architecture devoid of any cell nucleus. ECM proteins, such as collagen, elastin, and glycosaminoglycans were present even after detergentenzymatic decellularization. Immunohistochemical analysis of acellular scaffold showed weak expression of Gal 1, 3 Gal epitope compared with native tissue. For performing recellularization, human amnion-derived mesenchymal stem cells (MSCs) and epithelial cells were seeded onto acellular esophagus in a perfusion-rotation bioreactor. In recellularized esophagus, immunohistochemistry showed infiltration of MSCs from adventitia into the muscularis externa and differentiation of MSCs into the smooth muscle actin and few endothelial cells (CD31). Our study demonstrates successful preparation and characterization of a decellularized esophagus with reduced load of Gal 1, 3 Gal epitope with preserved architecture and ECM proteins similar to native tissue. Upon subsequent recellularization, xenogeneic acellular esophagus also supported stem cell growth and partial differentiation of stem cells. Hence, the current study offers the hope for preparing a tissue-engineered esophagus in vitro which can be transplanted further into pigs for further in vivo evaluation.
In vitro andin vivo proposal of an artificial esophagus
Journal of Biomedical Materials Research Part A, 2006
Artificial materials and autologous tissues used for esophageal reconstruction often induce complications like stenosis and leakage at long-term follow-up. This study evaluates the possibility to obtain in vitro an implantable tissue-engineered esophagus composed of homologous esophageal acellular matrix and autologous smooth muscle cells (SMCs). Acellular matrices obtained by detergent-enzymatic method did not present any major histocompatibility complex marker and expressed bFGF as protein, showing angiogenic activity in vivo on the chick embryo chorioallantoic membrane (CAM). Moreover, they supported cell adhesion, and inasmuch as just after 24 h from seeding, the scaffold appeared completely covered by SMCs. To verify the biocompatibility of our constructs, defects created in the porcine esophageal wall were covered using homologous acellular matrices with and without cultures of autologous SMCs. At 3 week from surgery, the patches composed of only acellular matrices showed a more severe inflammatory response and were negative for α-smooth muscle actin immunostaining. In contrast, the cell-matrix implants presented ingrowth of SMCs, showing an early organization into small fascicules. Collectively, these results suggest that patches composed of homologous esophageal acellular matrix and autologous SMCs may represent a promising tissue-engineering approach for the repair of esophageal injuries. © 2006 Wiley Periodicals, Inc. J Biomed Mater Res, 2006
A narrative review of esophageal tissue engineering and replacement: where are we?
Annals of Translational Medicine
Long-gap esophageal defects, whether congenital or acquired, are very difficult to manage. Any significant surgical peri-esophageal dissection that is performed to allow for potential stretching of two ends of a defect interrupts the esophageal blood supply and leads to complications such as leak and stricture, even in the youngest, healthiest patients. The term "congenital" applied to these defects refers mainly to long-gap esophageal atresia (LGA). Causes of acquired long-segment esophageal disruption include recurrent leaks and fistulae after primary repair, refractory GERD, caustic ingestions, cancer, and strictures. 5,000-10,000 patients per year in the US require esophageal replacement. Gastric, colonic, and jejunal pull-up surgeries are fraught with high rates of both short and long term complications thus creating a space for a better option. Since the 1970's many groups around the world have been unsuccessfully attempting esophageal replacement with tissue-engineered grafts in various animal models. But, recent advances in these models are now combining novel technologic advances in materials bioscience, stem-cell therapies, and transplantation and are showing increasing promise to human translational application. Transplantation has been heretofore unsuccessful, but given modern improvements in transplant microsurgery and immunosuppressive medications, pioneering trials in animal models are being undertaken now. These rapidly evolving medical innovations will be reviewed here.