a-reliable-porcine-fascio-cutaneous-flap-model-for-vascularized

A Reliable Porcine Fascio-Cutaneous Flap Model for Vascularized Composite Allografts Bioengineering Studies

Vascularized Composite Allografts (VCA) such as hand, face, or penile transplant represents the cutting-edge treatment for devastating skin defects, ...

establishing-swine-model-to-study-uterus-dynamic-preservation

Establishing a Swine Model to Study Uterus Dynamic Preservation and Transplantation

To date, uterus transplantation is the only option for women with absolute uterine infertility, such as those with Rokitansky syndrome, to experience ...

Massachusetts General Hospital, Harvard Medical School

Shriners Hospital for Children

Shriners Children's Boston

Cedars-Sinai Hospital

Modern microsurgical techniques have made possible a broad spectrum of novel means for the reconstruction of complex bone and soft tissue defects. These techniques, in combination with developments in transplant immunology, have led to successful hand and facial allotransplantation and achievement of the highest rung in the reconstructive ladder - truly replacing like with like. The utilization of contemporary microsurgical technique in the context of vascularized composite allotransplantation (VCA) (1) permits successful technical execution and feasibility of VCA, (2) facilitates the study of immunologic tolerance in VCA preclinical models, and (3) optimizes functional VCA outcomes.

The Advent of Vascularized Composite Allotransplantation.

Infection with SARS-CoV-2 is responsible for the COVID-19 crisis affecting the whole world. This virus can provoke acute respiratory distress syndrome (ARDS) leading to overcrowed the intensive care unit (ICU). Over the last months, worldwide experience demonstrated that the ARDS in COVID-19 patients are in many ways "atypical". The mortality rate in ventilated patients is high despite the application of the gold standard treatment (protective ventilation, curare, prone position, inhaled NO). Several studies suggested that the SARS-CoV-2 could interact negatively on red blood cell homeostasis. Furthermore, SarsCov2 creates Reactive Oxygen Species (ROS), which are toxic and generate endothelial dysfunction. Hypothesis/objective(s) We hypothesis that HEMO2Life® administrated intravenously is safe and could help symptomatically the patient condition. It would increase arterial oxygen content despite lung failure and allow better tissue oxygenation control. The use of HEMO2Life® is also interesting due to its anti-oxidative effect preventing cytokine storm induced by the SARS-CoV-2. Evaluation of the hypothesis: Hemarina is based on the properties of the hemoglobin of the Arenicola marina sea-worm (HEMO2Life®). This extracellular hemoglobin has an oxygen capacity 40 times greater than the hemoglobin of vertebrates. Furthermore, the size of this molecule is 250 times smaller than a human red blood cell, allowing it to diffuse in all areas of the microcirculation, without diffusing outside the vascular sector. It possesses an antioxidative property du a Superoxide Dismutase Activity. This technology has been the subject of numerous publications and HEMO2Life® was found to be well-tolerated and did not induce toxicity. It was administered intravenously to hamsters and rats, and showed no acute effect on heart rate and blood pressure and did not cause microvascular vasoconstriction. In preclinical in vivo models (mice, rats, and dogs), HEMO2Life® has enabled better tissue oxygenation, especially in the brain. This molecule has already been used in humans in organ preservation solutions and the patients showed no abnormal clinical signs.

Combating hypoxemia in COVID-19 patients with a natural oxygen carrier, HEMOLife® (M101).

The gold standard in nipple reconstruction remains the autologous skin flap. Unfortunately the results are not satisfying with up to 75% loss of nipple projection over time.1-8 Existing studies investigated the use of primates as a source of implants.9,10 We hypothesized that porcine nipple can serve as a perfect shape-supporting implant due to functional similarities to human nipple.11,12 Our group developed a decellularization protocol to obtain an acellular nipple scaffold (ANS) for nipple reconstruction.

Acellular nipple scaffold development, characterization, and preliminary biocompatibility assessment in a swine model.

 Blood perfusion quality of a flap is the main prognostic factor for success. Microvascular evaluation remains mostly inaccessible. We aimed to evaluate the microflow imaging mode, MV-Flow, in assessing flap microvascularization in a pig model of the fascio-cutaneous flap.  On five pigs, bilateral saphenous fascio-cutaneous flaps were procured on the superficial femoral vessels. A conventional ultrasound evaluation in pulsed Doppler and color Doppler was conducted on the ten flaps allowing for the calculation of the saphenous artery flow rate. The MV-Flow mode was then applied: for qualitative analysis, with identification of saphenous artery collaterals; then quantitative, with repeated measurements of the Vascularity Index (VI), percentage of pixels where flow is detected relative to the total ultrasound view area. The measurements were then repeated after increasing arterial flow by clamping the distal femoral artery.  The MV-Flow mode allowed a better follow-up of the saphenous artery's collaterals and detected microflows not seen with the color Doppler. The VI was correlated to the saphenous artery flow rate (Spearman rho of 0.64;  = 0.002) and allowed to monitor the flap perfusion variations.  Ultrasound imaging of microvascularization by MV-Flow mode and its quantification by VI provides valuable information in evaluating the microvascularization of flaps.

Microvascular assessment of fascio-cutaneous flaps by ultrasound: A large animal study.

Machine perfusion has developed rapidly since its first use in solid organ transplantation. Likewise, reconstructive surgery has kept pace, and ex vivo perfusion appears as a new trend in vascularized composite allotransplants preservation. In autologous reconstruction, fasciocutaneous flaps are now the gold standard due to their low morbidity (muscle sparing) and favorable functional and cosmetic results. However, failures still occasionally arise due to difficulties encountered with the vessels during free flap transfer. The development of machine perfusion procedures would make it possible to temporarily substitute or even avoid microsurgical anastomoses in certain complex cases. We performed oxygenated acellular sub-normothermic perfusions of fasciocutaneous flaps for 24 and 48 h in a porcine model and compared continuous and intermittent perfusion regimens. The monitored metrics included vascular resistance, edema, arteriovenous oxygen gas differentials, and metabolic parameters. A final histological assessment was performed. Porcine flaps which underwent successful oxygenated perfusion showed minimal or no signs of cell necrosis at the end of the perfusion. Intermittent perfusion allowed overall better results to be obtained at 24 h and extended perfusion duration. This work provides a strong foundation for further research and could lead to new and reliable reconstructive techniques.

Towards Optimizing Sub-Normothermic Machine Perfusion in Fasciocutaneous Flaps: A Large Animal Study.

Autonomization is a physiological process allowing a flap to develop  from the reconstructed wound bed. This phenomenon has been used since the early application of flap surgeries but still remains poorly understood. Reconstructive strategies have greatly evolved since, and fasciocutaneous flaps have progressively replaced muscle-based reconstructions, ensuring better functional outcomes with great reliability. However, plastic surgeons still encounter challenges in complex cases where conventional flap reconstruction reaches its limitations. Furthermore, emerging bioengineering applications, such as decellularized scaffolds allowing a complex extracellular matrix to be repopulated with autologous cells, also face the complexity of revascularization. The objective of this article is to gather evidence of autonomization phenomena. A systematic review of flap autonomization is then performed to document the minimum delay allowing this process. Finally, past and potential applications in bio- and tissue-engineering approaches are discussed, highlighting the potential for in vivo revascularization of acellular scaffolds.

The Autonomization Principle in Vascularized Flaps: An Alternative Strategy for Composite Tissue Scaffold In Vivo Revascularization.

Mastectomy is a primary treatment for breast cancer patients, and both autologous and implant-based reconstructive techniques have shown excellent results. In recent years, advancements in bioengineering have led to a proliferation of innovative approaches to breast reconstruction. This article comprehensively explores the promising perspectives offered by bioengineering and tissue engineering in the field of breast reconstruction.

Tissue engineering strategies for breast reconstruction: a literature review of current advances and future directions.

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.

Optimized Decellularization of a Porcine Fasciocutaneaous Flap.

Ex-vivo preservation of transplanted organs is undergoing spectacular advances. Machine perfusion is now used in common practice for abdominal and thoracic organ transportation and preservation, and early results are in favor of substantially improved outcomes. It is based on decreasing ischemia-reperfusion phenomena by providing physiological or sub-physiological conditions until transplantation. Alternatively, supercooling techniques involving static preservation at negative temperatures while avoiding ice formation have shown encouraging results in solid organs. Here, the rationale is to decrease the organ's metabolism and need for oxygen and nutrients, allowing for extended preservation durations. The aim of this work is to review all advances of supercooling in transplantation, browsing the literature for each organ. A specific objective was also to study the initial evidence, the prospects, and potential applications of supercooling preservation in Vascularized Composite Allotransplantation (VCA). This complex entity needs a substantial effort to improve long-term outcomes, marked by chronic rejection. Improving preservation techniques is critical to ensure the favorable evolution of VCAs, and supercooling techniques could greatly participate in these advances.

Supercooling: A Promising Technique for Prolonged Organ Preservation in Solid Organ Transplantation, and Early Perspectives in Vascularized Composite Allografts.

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.

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

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 (HS), 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 ( = 0.483) but was reduced with the sodium iodide treatment ( = 0.007). In the MISMATCH group, survival was similar between the saline and hydrogen sulfide treatments ( = 0.483) but decreased with the sodium iodide treatment ( = 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.

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

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  for 24 h with either SNMP ( = 3) or SCS ( = 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.

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

Vascularized composite allotransplantations are complex procedures with substantial functional impact on patients. Extended preservation of VCAs is of major importance in advancing this field. It would result in improved donor-recipient matching as well as the potential for ex vivo manipulation with gene and cell therapies. Moreover, it would make logistically feasible immune tolerance induction protocols through mixed chimerism. Supercooling techniques have shown promising results in multi-day liver preservation. It consists of reaching sub-zero temperatures while preventing ice formation within the graft by using various cryoprotective agents. By drastically decreasing the cell metabolism and need for oxygen and nutrients, supercooling allows extended preservation and recovery with lower ischemia-reperfusion injuries. This study is the first to demonstrate the supercooling of a large animal model of VCA. Porcine hindlimbs underwent 48 h of preservation at - 5 °C followed by recovery and normothermic machine perfusion assessment, with no issues in ice formation and favorable levels of injury markers. Our findings provide valuable preliminary results, suggesting a promising future for extended VCA preservation.

ArticleTotal : 2	Year
Publication title Cited by 7	2022
Publication title Cited by 3	2024

Article	Year
The Advent of Vascularized Composite Allotransplantation. Clinics in plastic surgery\| PubMed ID: 28340673	2017
Combating hypoxemia in COVID-19 patients with a natural oxygen carrier, HEMOLife® (M101). Medical hypotheses\| PubMed ID: 33308935	2021
Acellular nipple scaffold development, characterization, and preliminary biocompatibility assessment in a swine model. Plastic and reconstructive surgery\| PubMed ID: 36472499	2022
Microvascular assessment of fascio-cutaneous flaps by ultrasound: A large animal study. Frontiers in physiology\| PubMed ID: 36589429	2022
Towards Optimizing Sub-Normothermic Machine Perfusion in Fasciocutaneous Flaps: A Large Animal Study. Bioengineering (Basel, Switzerland)\| PubMed ID: 38136006	2023
The Autonomization Principle in Vascularized Flaps: An Alternative Strategy for Composite Tissue Scaffold In Vivo Revascularization. Bioengineering (Basel, Switzerland)\| PubMed ID: 38136031	2023
Tissue engineering strategies for breast reconstruction: a literature review of current advances and future directions. Annals of translational medicine\| PubMed ID: 38304901	2024
Optimized Decellularization of a Porcine Fasciocutaneaous Flap. Bioengineering (Basel, Switzerland)\| PubMed ID: 38671744	2024
Supercooling: A Promising Technique for Prolonged Organ Preservation in Solid Organ Transplantation, and Early Perspectives in Vascularized Composite Allografts. Frontiers in transplantation\| PubMed ID: 38682043	2023
Effect of Subnormothermic Machine Perfusion on the Preservation of Vascularized Composite Allografts After Prolonged Warm Ischemia. Transplantation\| PubMed ID: 38722685	2024
Exploring Iodide and Hydrogen Sulfide as ROS Scavengers to Delay Acute Rejection in MHC-Defined Vascularized Composite Allografts. Antioxidants (Basel, Switzerland)\| PubMed ID: 38790636	2024
Machine Perfusion Enables 24-h Preservation of Vascularized Composite Allografts in a Swine Model of Allotransplantation. Transplant international : official journal of the European Society for Organ Transplantation\| PubMed ID: 38813393	2024
VCA supercooling in a swine partial hindlimb model. Scientific reports\| PubMed ID: 38824189	2024

Curtis L. Cetrulo

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