Machine Perfusion Enables 24-h Preservation of Vascularized Composite Allografts in a Swine Model of Allotransplantation.

The current gold standard for preserving vascularized composite allografts (VCA) is 4°C static cold storage (SCS), albeit muscle vulnerability to ischemia can be described as early as after 2 h of SCS. Alternatively, machine perfusion (MP) is growing in the world of organ preservation. Herein, we investigated the outcomes of oxygenated acellular subnormothermic machine perfusion (SNMP) for 24-h VCA preservation before allotransplantation in a swine model. Six partial hindlimbs were procured on adult pigs and preserved ex vivo for 24 h with either SNMP (n = 3) or SCS (n = 3) before heterotopic allotransplantation. Recipient animals received immunosuppression and were followed up for 14 days. Clinical monitoring was carried out twice daily, and graft biopsies and blood samples were regularly collected. Two blinded pathologists assessed skin and muscle samples. Overall survival was higher in the SNMP group. Early euthanasia of 2 animals in the SCS group was linked to significant graft degeneration. Analyses of the grafts showed massive muscle degeneration in the SCS group and a normal aspect in the SNMP group 2 weeks after allotransplantation. Therefore, this 24-h SNMP protocol using a modified Steen solution generated better clinical and histological outcomes in allotransplantation when compared to time-matched SCS.

Effect of Subnormothermic Machine Perfusion on the Preservation of Vascularized Composite Allografts After Prolonged Warm Ischemia.

BACKGROUND: Warm ischemia time (WIT) and ischemia-reperfusion injury are limiting factors for vascularized composite allograft (VCA) transplantation. Subnormothermic machine perfusion (SNMP) has demonstrated the potential to extend WIT in organ transplantation. This study evaluates the effect of SNMP on VCA viability after prolonged WIT. METHODS: Rat hindlimbs underwent WIT for 30, 45, 60, 120, 150, or 210 min, followed by 3-h SNMP. Monitoring of perfusion parameters and outflow determined the maximum WIT compatible with limb viability after SNMP. Thereafter, 2 groups were assessed: a control group with inbred transplantation (Txp) after 120 min of WIT and an experimental group that underwent WIT + SNMP + Txp. Graft appearance, blood gas, cytokine levels, and histology were assessed for 21 d. RESULTS: Based on potassium levels, the limit of WIT compatible with limb viability after SNMP is 120 min. Before this limit, SNMP reduces potassium and lactate levels of WIT grafts to the same level as fresh grafts. In vivo, the control group presented 80% graft necrosis, whereas the experimental group showed no necrosis, had better healing (P = 0.0004), and reduced histological muscle injury (P = 0.012). Results of blood analysis revealed lower lactate, potassium levels, and calcium levels (P = 0.048) in the experimental group. Both groups presented an increase in interleukin (IL)-10 and IL-1b/IL-1F2 with a return to baseline after 7 to 14 d. CONCLUSIONS: Our study establishes the limit of WIT compatible with VCA viability and demonstrates the effectiveness of SNMP in restoring a graft after WIT ex vivo and in vivo, locally and systemically.

Intercellular transfer of MHC molecules in T cell alloimmunity and allotransplantation.

After transplantation of allogeneic tissues and organs, recognition by recipient T cells of donor MHC molecules initiates the pro-inflammatory adaptive immune response leading to allograft rejection. T cell allorecognition has long been known to be mediated via two distinct pathways: the direct pathway in which T cells recognize intact allogeneic MHC molecules displayed on donor cells and the indirect pathway whereby T cells recognize donor MHC peptides processed and presented by recipient antigen-presenting cells (APCs). It is believed that direct allorecognition is the driving force behind early acute allograft rejection while indirect allorecognition is involved in chronic allograft rejection, a progressive condition characterized by graft vasculopathy and tissue fibrosis. Recently, we and others have reported that after transplantation of allogeneic skin and organs, donor MHC molecules are transferred from donor cells to the host's APCs via trogocytosis or extracellular vesicles. Recipient APCs having captured donor MHC molecules can either present them to T cells in the intact form on their surface (semi-direct pathway) or the form of peptides bound to self-MHC molecules (indirect pathway). The present article provides an overview of recent studies evaluating the role of intercellular exchange of MHC molecules in T cell alloimmunity and its contribution to allograft rejection and tolerance.

Exploring Iodide and Hydrogen Sulfide as ROS Scavengers to Delay Acute Rejection in MHC-Defined Vascularized Composite Allografts.

Vascularized composite allografts (VCA) face ischemic challenges due to their limited availability. Reperfusion following ischemia triggers oxidative stress and immune reactions, and scavenger molecules could mitigate ischemia-reperfusion injuries and, therefore, immune rejection. We compared two scavengers in a myocutaneous flap VCA model. In total, 18 myocutaneous flap transplants were performed in Major histocompatibility complex (MHC)-defined miniature swine. In the MATCH group (n = 9), donors and recipients had minor antigen mismatch, while the animals were fully mismatched in the MISMATCH group (n = 9). Grafts were pretreated with saline, sodium iodide (NaI), or hydrogen sulfide (H2S), stored at 4 °C for 3 h, and then transplanted. Flaps were monitored until clinical rejection without immunosuppression. In the MATCH group, flap survival did not significantly differ between the saline and hydrogen sulfide treatments (p = 0.483) but was reduced with the sodium iodide treatment (p = 0.007). In the MISMATCH group, survival was similar between the saline and hydrogen sulfide treatments (p = 0.483) but decreased with the sodium iodide treatment (p = 0.007). Rhabdomyolysis markers showed lower but non-significant levels in the experimental subgroups for both the MATCH and MISMATCH animals. This study provides insightful data for the field of antioxidant-based approaches in VCA and transplantation.

Acute Rejection Rates in Vascularized Composite Allografts: A Systematic Review of Case Reports.

INTRODUCTION: Vascularized Composite Allografts (VCA) are usually performed in a full major histocompatibility complex mismatch setting, with a risk of acute rejection depending on factors such as the type of immunosuppression therapy and the quality of graft preservation. In this systematic review, we present the different immunosuppression protocols used in VCA and point out relationships between acute rejection rates and possible factors that might influence it. METHODS: This systematic review was performed according to the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines. We systematically searched Medline (PubMed), Embase, and The Cochrane Library between November 2022 and February 2023, using following Mesh Terms: Transplant, Transplantation, Hand, Face, Uterus, Penis, Abdominal Wall, Larynx, and Composite Tissue Allografts. All VCA case reports and reviews describing multiple case reports were included. RESULTS: We discovered 211 VCA cases reported. The preferred treatment was a combination of antithymocyte globulins, mycophenolate mofetil (MMF), tacrolimus, and steroids; and a combination of MMF, tacrolimus, and steroids for induction and maintenance treatment, respectively. Burn patients showed a higher acute rejection rate (P = 0.073) and were administered higher MMF doses (P = 0.020). CONCLUSIONS: In contrast to previous statements, the field of VCA is not rapidly evolving, as it has encountered challenges in addressing immune-related concerns. This is highlighted by the absence of a standardized immunosuppression regimen. Consequently, more substantial data are required to draw more conclusive results regarding the immunogenicity of VCAs and the potential superiority of one immunosuppressive treatment over another. Future efforts should be made to report the VCA surgeries comprehensively, and muti-institutional long-term prospective follow-up studies should be performed to compare the number of acute rejections with influencing factors.

Optimized Decellularization of a Porcine Fasciocutaneaous Flap.

Reconstructive techniques to repair severe tissue defects include the use of autologous fasciocutaneous flaps, which may be limited due to donor site availability or lead to complications such as donor site morbidity. A number of synthetic or natural dermal substitutes are in use clinically, but none have the architectural complexity needed to reconstruct deep tissue defects. The perfusion decellularization of fasciocutaneous flaps is an emerging technique that yields a scaffold with the necessary composition and vascular microarchitecture and serves as an alternative to autologous flaps. In this study, we show the perfusion decellularization of porcine fasciocutaneous flaps using sodium dodecyl sulfate (SDS) at three different concentrations, and identify that 0.2% SDS results in a decellularized flap that is efficiently cleared of its cellular material at 86%, has maintained its collagen and glycosaminoglycan content, and preserved its microvasculature architecture. We further demonstrate that the decellularized graft has the porous structure and growth factors that would facilitate repopulation with cells. Finally, we show the biocompatibility of the decellularized flap using human dermal fibroblasts, with cells migrating as deep as 150 µm into the tissue over a 7-day culture period. Overall, our results demonstrate the promise of decellularized porcine flaps as an interesting alternative for reconstructing complex soft tissue defects, circumventing the limitations of autologous skin flaps.

Modified Tail Vein and Penile Vein Puncture for Blood Sampling in the Rat Model.

Blood samples are required in most experimental animal designs to assess various hematological parameters. This paper presents two procedures for blood collection in rats: the lateral tail vein puncture and the dorsal penile vein puncture, which offer significant advantages over other previously described techniques. This study shows that these two procedures allow for fast sampling (under 10 min) and yield sufficient blood volumes for most assays (202 µL ± 67.7 µL). The dorsal penile vein puncture must be done under anesthesia, whereas the lateral tail vein puncture can be done on a conscious, restrained animal. Alternating these two techniques, therefore, enables blood draw in any situation. While it is always recommended for an operator to be assisted during a procedure to ensure animal welfare, these techniques require only a single operator, unlike most blood sampling methods that require two. Moreover, whereas these previously described methods (e.g., jugular stick, subclavian vein blood draw) require extensive prior training to avoid harm to or death of the animal, tail vein and dorsal penile vein puncture are rarely fatal. For all these reasons, and according to the context (e.g., for studies including male rats, during the perioperative or immediate postoperative period, for animals with thin tail veins), both techniques can be used alternately to enable repeated blood draws.

The Superficial Inferior Epigastric Artery Axial Flap to Study Ischemic Preconditioning Effects in a Rat Model.

Fasciocutaneous flaps (FCF) have become the gold standard for complex defect reconstruction in plastic and reconstructive surgery. This muscle-sparing technique allows transferring vascularized tissues to cover any large defect. FCF can be used as pedicled flaps or as free flaps; however, in the literature, failure rates for pedicled FCF and free FCF are above 5%, leaving room for improvement for these techniques and further knowledge expansion in this area. Ischemic preconditioning (I.P.) has been widely studied, but the mechanisms and the optimization of the I.P. regimen are yet to be determined. This phenomenon is indeed poorly explored in plastic and reconstructive surgery. Here, a surgical model is presented to study the I.P. regimen in a rat axial fasciocutaneous flap model, describing how to safely and reliably assess the effects of I.P. on flap survival. This article describes the complete surgical procedure, including suggestions to improve the reliability of this model. The objective is to provide researchers with a reproducible and reliable model to test various ischemic preconditioning regimens and assess their effects on flap survivability.

Pathways of Antigen Recognition by T Cells in Allograft Rejection.

The adaptive immune response leading to the rejection of allogeneic transplants is initiated and orchestrated by recipient T cells recognizing donor antigens. T-cell allorecognition is mediated via 3 distinct mechanisms: the direct pathway in which T cells recognize allogeneic major histocompatibility complex (MHC) molecules on donor cells, the indirect pathway through which T cells interact with donor peptides bound with self-MHC molecules on recipient antigen-presenting cells, and the recently described semidirect pathway whereby T cells recognize donor MHC proteins on recipient antigen-presenting cells. In this article, we present a description of each of these allorecognition pathways and discuss their role in acute and chronic rejection of allogeneic transplants.