Immunogenicity and tolerance induction in vascularized composite allotransplantation

Vascularized composite allotransplantation (VCA) is the transplantation of multiple tissues such as skin, muscle, bone, nerve, and vessels, as a functional unit (i.e., hand or face) to patients suffering from major tissue trauma and functional deficits. Though the surgical feasibility has been optimized, issues regarding graft rejection remains. VCA rejection involves a diverse population of cells but is primarily driven by both donor and recipient lymphocytes, antigen-presenting cells, macrophages, and other immune as well as donor-derived cells. In addition, it is commonly understood that different tissues within VCA, such as the skin, elicits a stronger rejection response. Currently, VCA recipients are required to follow potent and lifelong immunosuppressing regimens to maximize graft survival. This puts patients at risk for malignancies, opportunistic infections, and cancers, thereby posing a need for less perilous methods of inducing graft tolerance. This review will provide an overview of cell populations and mechanisms, specific tissue involved in VCA rejection, as well as an updated scope of current methods of tolerance induction

Review of machine perfusion studies in vascularized composite allotransplant preservation

The applications of Vascularized composite allotransplantation (VCA) are increasing since the first successful hand transplantation in 1998. However, the abundance of muscle tissue makes VCA's vulnerable to ischemia-reperfusion injury (IRI), which has detrimental effects on the outcome of the procedure, restricting allowable donor-to-recipient time and limiting its widespread use. The current clinical method is Static cold storage (SCS) and this allows only 6 h before irreversible damage occurs upon reperfusion. In order to overcome this obstacle, the focus of research has been shifted towards the prospect of ex-vivo perfusion preservation which already has an established clinical role in solid organ transplants especially in the last decade. In this comprehensive qualitative review, we compile the literature on all VCA machine perfusion models and we aim to highlight the essentials of an ex vivo perfusion set-up, the different strategies, and their associated outcomes

Advances in Ischemia Reperfusion Injury Prevention in Free Flaps and Vascularized Composite Allotransplantation

In Plastic and Reconstructive Surgery, ischemia reperfusion injury (IRI) prevention is of utmost importance in free flaps and vascularized composite allotransplantation (VCA) to continue increasing accessibility to these advanced reconstructive options. At present, free flaps and VCA undergo irreversible ischemic damage at 3 hours due to the highly metabolic nature of skeletal muscle, and static cold storage (SCS) can only extend this to 4–6 hours. It is important to understand that one of the major challenges with transplanting composite tissues is that each tissue has a unique tolerance and mechanism to ischemia-reperfusion. Research targeting attenuation of IRI can be subdivided into 3 time periods: the pre-ischemic, ischemic, and post-ischemic. In the pre-ischemic period, there are conditioning methods, the delay phenomenon, which is already used clinically, pharmacologic, and stem cell strategies. In the ischemic period, SCS is used clinically, whilst other preservation methods including cryopreservation, vitrification, machine perfusion, and pharmacologic strategies are being studied. Lastly, in the post-ischemic period, our greatest clinical tool is close post-operative monitoring, however conditioning methods, and pharmacologic strategies have been studied. This chapter covers IRI in tissues implicated in free flaps and VCA, and several prevention strategies either currently in use or in pre-clinical studies

De-Epithelialization Protocol with Tapered Sodium Dodecyl Sulfate Concentrations Enhances Short-Term Chondrocyte Survival in Porcine Chimeric Tracheal Allografts

Background: Tracheal transplantation is indicated in cases where injury exceeds 50% of the organ in adults and 30% in children. However, transplantation is not yet considered a viable treatment option partly due to high morbidity and mortality associated with graft rejection. Recently, decellularization (decell) has been explored as a technique for creating bioengineered tracheal grafts. However, risk of post-operative stenosis increases due to the death of chondrocytes, which are critical to maintain the biochemical and mechanical integrity of tracheal cartilage. In this project, we propose a new de-epithelialization protocol that adequately removes epithelial, mucosal, and submucosal cells while maintaining a greater proportion of viable chondrocytes. Methods: The trachea of adult male outbred Yorkshire pigs were extracted, decontaminated, and decellularized according to the original and new protocols before incubation at 37 °C in DMEM for 10 days. Chondrocyte viability was quantified immediately following post-decellularization and on days 1, 4, 7, and 10. Histology was performed pre-decellularization, post-decellularization, and post-incubation. Results: The new protocol showed a significant (p < 0.05) increase in chondrocyte viability up to four days after de-ep when compared to the original protocol. We also found that the new protocol preserves ECM composition to a similar degree as the original protocol. When scaffolds created using the new protocol were re-epithelialized, cell growth curves were near identical to published data from the original protocol. Conclusion: While not without limitations, our new protocol may be used to engineer chimeric tracheal allografts without the need for cartilage regeneration

Advances in Tracheal Tissue-Engineering: Evaluation of the Structural Integrity, Immunogenicity and Recellularization of a Decellularized Circumferential Long-segment Trachea for Airway Transplantation

Subglottic stenosis, malignancy and traumatic injury to the trachea require surgical resection. When defects are less than 50% of the tracheal length in adults and 1/3 in children, a circumferential resection and primary anastomosis affords excellent results. For longer lesions, on the other hand, there are no currently acceptable solutions leading to patients requiring permanent tracheostomies or palliative treatment. Tracheal replacement approaches with synthetic prosthesis and scaffolds have all led to inflammation, obstruction, mucous build-up and eventual restenosis. Tissue-engineering approaches using recipients’ own stem cells and biologic scaffolds derived from decellularized donor trachea have shown great promise. They have the potential to abrogate the need for immunosuppressive therapy. Our research focuses on three major limitations in this field including the structural integrity, the immunogenicity and the recellularization of donor tracheae. We compared three decellularization protocols, quantified and qualified the extracellular matrix (ECM) components and performed compliance measurements on large circumferential tracheal scaffolds following cyclical decellularization techniques and illustrated significant differences in ECM composition and resultant structural integrity of decellularized tracheal scaffolds depending on the protocol. In addition, we investigated the immunogenicity of decellularized and recellularized tracheal allografts at a protein level and in vitro and in vivo T cell proliferation. Decellularization is associated with a delay in leukocyte infiltration and recellularization promoted cartilage preservation and the recruitment of regulatory T cells. We described a dramatic increase of TGF-β1 in recellularized scaffolds. Moreover, we designed a dual-chamber bioreactor for recellularization of tracheal allografts. Our method allowed for dynamic perfusion seeding, confirmed adherence of two different cell types and achieved higher cell numbers and homogeneous structures compared to traditional static seeding methods. In summary, we have identified and addressed three major limitations for tissue-engineering of long-segment decellularized tracheal scaffolds relating to structural integrity, immunogenicity and recellularization techniques.Ph

Adipose-Derived Stem Cells: Angiogenetic Potential and Utility in Tissue Engineering

Adipose tissue (AT) is a large and important energy storage organ as well as an endocrine organ with a critical role in many processes. Additionally, AT is an enormous and easily accessible source of multipotent cell types used in our day for all types of tissue regeneration. The ability of adipose-derived stem cells (ADSCs) to differentiate into other types of cells, such as endothelial cells (ECs), vascular smooth muscle cells, or cardiomyocytes, is used in tissue engineering in order to promote/stimulate the process of angiogenesis. Being a key for future successful clinical applications, functional vascular networks in engineered tissue are targeted by numerous in vivo and ex vivo studies. The article reviews the angiogenic potential of ADSCs and explores their capacity in the field of tissue engineering (TE)

Advances in Tracheal Reconstruction

Summary: A recent revival of global interest for reconstruction of long-segment tracheal defects, which represents one of the most interesting and complex problems in head and neck and thoracic reconstructive surgery, has been witnessed. The trachea functions as a conduit for air, and its subunits including the epithelial layer, hyaline cartilage, and segmental blood supply make it particularly challenging to reconstruct. A myriad of attempts at replacing the trachea have been described. These along with the anatomy, indications, and approaches including microsurgical tracheal reconstruction will be reviewed. Novel techniques such as tissue-engineering approaches will also be discussed. Multiple attempts at replacing the trachea with synthetic scaffolds have been met with failure. The main lesson learned from such failures is that the trachea must not be treated as a “simple tube.” Understanding the anatomy, developmental biology, physiology, and diseases affecting the trachea are required for solving this problem

Computational fluid dynamics for enhanced tracheal bioreactor design and long-segment graft recellularization

Abstract Successful re-epithelialization of de-epithelialized tracheal scaffolds remains a challenge for tracheal graft success. Currently, the lack of understanding of the bioreactor hydrodynamic environment, and its relation to cell seeding outcomes, serve as major obstacles to obtaining viable tracheal grafts. In this work, we used computational fluid dynamics to (a) re-design the fluid delivery system of a trachea bioreactor to promote a spatially uniform hydrodynamic environment, and (b) improve the perfusion cell seeding protocol to promote homogeneous cell deposition. Lagrangian particle-tracking simulations showed that low rates of rotation provide more uniform circumferential and longitudinal patterns of cell deposition, while higher rates of rotation only improve circumferential uniformity but bias cell deposition proximally. Validation experiments with human bronchial epithelial cells confirm that the model accurately predicts cell deposition in low shear stress environments. We used the acquired knowledge from our particle tracking model, as a guide for long-term tracheal repopulation studies. Cell repopulation using conditions resulting in low wall shear stress enabled enhanced re-epithelialization of long segment tracheal grafts. While our work focuses on tracheal regeneration, lessons learned in this study, can be applied to culturing of any tissue engineered tubular scaffold