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, , , , , , Pediatric Surgery Unit, General Surgery Department, Faculty of Medicine, Al-Azhar University (Assiut Branch), Assiut, Egypt
Received 18 August 2023, Revised 26 March 2024, Accepted 5 April 2024, Available online 16 April 2024.
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Pediatric surgeons have faced esophageal reconstruction challenges for decades owing to a variety of congenital and acquired conditions. This work aimed to introduce a reproducible and efficient approach for creating tissue-engineered esophageal tissue using bone marrow mesenchymal stem cells (BMSCs) cultured in preconditioned mediums seeded on a sheep decellularized tunica vaginalis (DTV) scaffold for partial reconstruction of a rabbit’s esophagus.
DTV was performed using SDS and Triton X-100 solutions. The decellularized grafts were employed alone (DTV group) or after recellularization with BMSCs cultured for 10 days in preconditioned mediums (RTV group) for reconstructing a 3 cm segmental defect in the cervical esophagus of rabbits (n = 20) after the decellularization process was confirmed. Rabbits were observed for one month, after which they were euthanized, and the reconstructed esophagi were harvested for histological analysis.
Six rabbits in the DTV group and eight rabbits in the RTV group survived until the end of the one-month study period. Despite histological examination demonstrating that both grafts completely repaired the esophageal defect, the RTV graft demonstrated a histological structure similar to that of the normal esophagus. The reconstructed esophagi in the RTV group revealed the arrangement of the different layers of the esophageal wall with the formation of newly formed blood vessels and Schwann-like cells.
DTV xenograft is a novel scaffold that promotes cell adhesion and differentiation and might be effectively utilized for regenerating esophageal tissue, paving the way for future clinical trials in pediatric patients.
The esophagus is a complex organ with poor regenerative capability, rendering regeneration challenging [1]. Congenital or acquired, long-gap esophageal defects are life-threatening and dramatically affect the quality of life [1]. The predominant indication in pediatric patients is long-gap esophageal atresia [2]. Other frequent causes of esophageal replacement in children include recurrent esophageal atresia or tracheoesophageal fistula leaks following primary repair, corrosive strictures, foreign body impaction, and button battery erosions, in addition to several malignancies [3].
Current medical procedures such as gastric, colonic, and jejunal pull-up operations have high risks of both short- and long-term morbidity [2]. Allotransplant is not an appropriate treatment option for children because of the shortage of organs of the proper size, the risk of rejection, and the probable lifetime immunosuppression necessary [4,5]. Therefore, the prospect of esophageal replacement using tissue-engineered grafts has been introduced as an alternative to the high morbidity of replacement surgeries [6].
A tissue-engineering approach, in which a transplantable conduit is bioengineered to replace the resected segment, avoiding the need to harvest replacement tissues from the patient’s own body, would avoid high-risk surgeries, be readily available, and likely reduce surgery-related mortality and morbidity while improving long-term functional outcome [7]. Therefore, tissue engineering has offered a new promise to imitate the native microenvironment and create tubular scaffolds to deal with the partial and full-length defects of the esophagus [1]. The most difficult aspects of esophageal regeneration are the need for a suitable scaffold, the required cell type, vascularization, and a suitable microenvironment [8]. To date, several scaffolds have been investigated as possible replacements for the esophagus. However, success has yet to be achieved using these scaffolds [9,10]. Decellularized grafts are potential esophageal scaffolds that give the three-dimensional microarchitecture of natural tissue and may encourage cell development without causing inflammatory responses that result in graft stricture, leakage, or failure [2,11].
Tunica vaginalis communis is one of the proposed scaffolds, and it has demonstrated promising outcomes in the reconstruction of several organs [[12], [13], [14], [15], [16], [17]]. Tunica vaginalis has many advantages, including simplicity of harvesting, remarkable mechanical and physical qualities, a lack of antigenic properties, a lower foreign body response and wound infection, and use without treatment or preservation [16].
Many studies have reported that a cell-seeded scaffold has the greatest potential for a full tissue-engineered esophagus replacement [[18], [19], [20]]. Mesenchymal stem cells (MSCs) continue to be an attractive source for cellular seeding owing to their pluripotency, ease of harvesting, and immunomodulatory effects [21]. Several in vivo esophageal studies have used MSC-seeded scaffolds [[22], [23], [24]]. MSCs stimulate both epithelialization and muscularization, which results in a more rapid and comprehensive generation of mucosal and muscular layers of the reconstructed esophagus with significant angiogenesis and reduced inflammation [21].
Although MSCs are believed to be a powerful and adaptable tool in cell therapy and tissue engineering applications, they have many limitations, including decreased survivability in culture or after transplantation and the probability of taking an undesired route of differentiation. To overcome those limitations, many studies have used scaffold-based MSC aggregates to achieve a 3D culture system that maintains a suitable niche and extracellular matrix, which in turn improves the viability and qualitative characteristics of the MSCs [25,26].
Moreover, in vitro preconditioning strategies of MSCs with a specific induction medium can increase the viability, proliferation, and paracrine properties of MSCs and therefore advance the therapeutic potential of the cells and their derived products, along with decreasing the probability of differentiation into unwanted cell lineages. We hypothesized that preconditioning MSCs with specific induction media to stimulate or at least initiate their differentiation into smooth muscle, endothelium, epithelium, and nerve cells could sufficiently repopulate a DTV scaffold and form the different cellular components of the esophageal wall, which would be superior to using the DTV alone in the reconstruction of an esophageal defect.
Therefore, the primary goal of this study was to assess the feasibility of reconstructing a 3-cm-long segmental defect in the cervical esophagus of a rabbit as a pediatric model using a xenogeneic sheep DTV graft. In addition, study the benefit of recellularization of DTV with autologous bone marrow mesenchymal stem cells (BMSCs) after seeding them in a preconditioned (neurogenic, endothelial, smooth muscle, and epithelial) induction medium to mimic the native esophageal environment.
The study was carried out at the Tissue Culture Unit, Histology Department, Faculty of Medicine, Ain Shams University and Department of Surgery, Anesthesiology and Radiology, Faculty of Veterinary Medicine, Suez Canal University. The Institutional Animal Use and Care Committee of the Faculty of Veterinary Medicine at Suez Canal University reviewed and authorized all experimental procedures (Approval Number 2021023).
Bone marrow was extracted from white New Zealand rabbits, and P2 or P3 cells were employed in subsequent experiments. BMSCs were identified as adherent cells capable of substantial proliferation, developing a fibroblast-like appearance, granular cytoplasm, many cytoplasmic processes, and a vesicular nucleus with multiple nucleoli. After 10–14 days of incubation, almost uniform populations of fibroblast-like cells were observed (Fig. S1). To validate cell identification, immunofluorescent
To the best of our knowledge, this study is the first to employ DTV as a decellularized scaffold for esophageal repair. The findings of this study have demonstrated that sheep DTV, a xenogeneic bioscaffold that had previously been used to augment dog bladders [12], could be used for esophageal repair. Whereas the seeded graft with BMSCs cultured in a different preconditioned medium for inducing differentiation led to the repair of the esophagus wall with nearly esophageal tissue morphology and
Taken together, the results suggest that the regenerative potential of DTV renders it a viable scaffold for esophageal repair. The use of a recellularized graft, including BMSCs cultured in vitro within a preconditioned medium, may promote epithelial, smooth muscle, and nervous tissue formation, decrease fibrosis, and improve angiogenesis. It has also shown a significant reduction in commonly seen complications and a substantial improvement in the regeneration of the esophagus wall, as
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Authors declare that they have no conflicts of interest to disclose.
In Memoriam of Prof. Dr. Abdel-Wahab Y. El-OKby, professor of pediatric surgery at the Faculty of Medicine at Al-Azhar University, Egypt. The authors would like to convey their sincere gratitude and appreciation for his keen supervision, continuous encouragement, and valuable direction during this work.
https://www.sciencedirect.com/science/article/abs/pii/S0022346824002501
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