Acellular Nipple Scaffold Development, Characterization, and Preliminary Biocompatibility Assessment in a Swine Model.

BACKGROUND: The 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. Existing studies investigated the use of primates as a source of implants. The authors hypothesized that the porcine nipple can serve as a perfect shape-supporting implant because of functional similarities to the human nipple. A decellularization protocol was developed to obtain an acellular nipple scaffold (ANS) for nipple reconstruction. METHODS: Tissue samples were collected from eight disease-free female Yorkshire pigs (60 to 70 kg) and then decellularized. The decellularization efficiency and extracellular matrix characterization was performed histologically and quantitatively (DNA, total collagen, elastin, and glycosaminoglycan content). In vitro and in vivo biocompatibility was determined by human dermal fibroblast culture and subcutaneous implantation of six ANSs in a single Yorkshire pig (60 to 70 kg), respectively. Inflammation and adverse events were monitored daily based on local clinical signs. RESULTS: The authors showed that all cellular structures and 96% of DNA [321.7 ± 57.6 ng DNA/mg wet tissue versus 11.7 ± 10.9 ng DNA/mg wet tissue, in native and ANS, respectively ( P < 0.001)] can be successfully removed. However, this was associated with a decrease in collagen [89.0 ± 11.4 and 58.8 ± 9.6 µg collagen/mg ( P < 0.001)] and elastin [14.2 ± 1.6 and 7.9 ± 2.4 µg elastin/mg ( P < 0.05)] and an increase in glycosaminoglycan content [5.0 ± 0.7 and 6.0 ± 0.8 ng/mg ( P < 0.05)]. ANS can support continuous cell growth in vitro and during preliminary biocompatibility tests in vivo. CONCLUSION: This is a preliminary report of a novel promising ANS for nipple reconstruction, but more research is needed to validate results. CLINICAL RELEVANCE STATEMENT: Breast cancer is very common among women. Treatment involves mastectomy, but its consequences affect patient mental well-being, and can lead to depression. Nipple-areola complex reconstruction is critical, and existing methods lead to unsatisfactory outcomes.

Tolerance of a Vascularized Composite Allograft Achieved in MHC Class-I-mismatch Swine via Mixed Chimerism.

Background: Vascularized composite allografts (VCAs) allow reconstruction of devastating injuries and amputations, yet require lifelong immunosuppression that is associated with significant morbidity. Induction of immune tolerance of VCAs would permit widespread use of these procedures. VCAs are acquired from deceased donors most likely to be fully-MHC-mismatched (in contrast to living-related renal transplant donor-recipient pairs matched at one MHC haplotype). After achieving VCA tolerance in a swine model equivalent to clinical living-related renal transplants (single-haplotype MHC mismatches: e.g., "mother-daughter"/haploidentical), we tested our protocol in MHC class I, class II, and fully-MHC-mismatched pairs. Although class II mismatched swine demonstrated similar results as the haploidentical scenario (stable mixed chimerism and tolerance), our protocol failed to prevent rejection of class I and full mismatch VCAs. Here, we describe a new adapted conditioning protocol that successfully achieved tolerance across MHC class-I-mismatch barriers in swine. Methods: Swine were treated with non-myeloablative total body and thymic irradiation two days prior to infusion of bone marrow cells from an MHC class I-mismatched donor. They also received a short-term treatment with CTLA4-Ig (Belatacept®) and anti-IL6R mAb (Tociluzimab®) and were transplanted with an osteomyocutaneous VCA from the same donor. Results: Stable mixed chimerism and tolerance of MHC class-I-mismatched VCAs was achieved in 3 recipients. Allograft tolerance was associated with a sustained lack of anti-donor T cell response and a concomitant expansion of double negative CD4-CD8- T cells producing IL-10. Conclusions: This study demonstrates the first successful mixed chimerism-induced VCA tolerance in a large animal model across a MHC class-I-mismatch. Future studies aimed at fully-mismatched donor-recipient pairs are under investigation with this protocol.

Treatment of hyperpigmentation after burn: A literature review.

OBJECTIFY: Skin pigmentation disorders are one of the most frequent sequelae after burn injury. While these conditions often improve over time, some are permanent and cause severe psychological disorders (especially on the face). Given the frequency of these disorders and their benign nature, the scientific community has great difficulty postponing these patient follow-ups. Publications on their management are rare, and there is no consensus on the gold standard treatment for skin dyschromia. Herein, we performed a literature review including the various treatments currently proposed to manage these hyperpigmentations. METHODS: All reported articles up to February 2021 were reviewed on Pubmed. Studies on the treatment of hyperpigmented scars were included if they were secondary to burn injuries. Excluded articles evaluated transient treatments, such as makeup, and articles on inflammatory hyperpigmentation without etiological details or not secondary to burns. RESULTS: 201 articles were identified, and 13 studies were included. Topical creams used in inflammatory hyperpigmented lesions such as hydroquinone and first-line retinoids are controversial due to their inconstant efficacy. Various types of laser and pulsed light treatments have shown their effectiveness but can also aggravate pigmentation. CONCLUSION: Dyschromia after burn remains a therapeutic challenge. Hyperpigmentations after burn should be treated on a case-by-case basis, using data from the literature, clinical experience and measuring the risk/benefit ratio.

Engineering Vascularized Composite Allografts Using Natural Scaffolds: A Systematic Review.

Vascularized composite allotransplantation (VCA) refers to the transplantation of multiple tissues as a functional unit from a deceased donor to a recipient with a severe injury. These grafts serve as potential replacements for traumatic tissue losses. The main problems are the consequences of the long immunosuppressive drugs and the lack of compatible donor. To avoid these limitations, decellularization/recellularization constitutes an attractive approach. The aim of decellularization/recellularization technology is to develop immunogenic free biological substitutes that will restore, maintain, or improve tissue and organ's function. A PubMed search was performed for articles on decellularization and recellularization of composite tissue allografts between February and March 2021, with no restrictions in publication year. The selected reports were evaluated in terms of decellularization protocols, assessment of decellularized grafts, and evaluation of their biocompatibility and repopulation with cells both in vitro and in vivo. The search resulted in a total of 88 articles. Each article was reviewed, 77 were excluded, and the remaining 11 articles reported decellularization of 12 different vascular composite allografts in humans (4), large animals (3), and small animals (rodents; 5). The decellularization protocol for VCA varies slightly between studies, but majority of the reports employ 1% sodium dodecyl sulfate as the main reagent for decellularization. The immunological response of the decellularized scaffolds remain poorly evaluated. Few authors have been able to attempt the recellularization and transplantation of these scaffolds. Successful transplantation seems to require prior recellularization. Decellularization/recellularization is a promising, growing, and emerging developing research field in vascular composite allotransplantation. Impact statement Tissue engineering for vascular composite allotransplantation using decellularization and recellularization approach is a fast-growing area of interest in the reconstructive surgery field. This review will be a very useful tool to get a clear overview for researchers interested in this field.

Optimization of Ex Vivo Machine Perfusion and Transplantation of Vascularized Composite Allografts.

BACKGROUND: Machine perfusion is gaining interest as an efficient method of tissue preservation of Vascularized Composite Allografts (VCA). The aim of this study was to develop a protocol for ex vivo subnormothermic oxygenated machine perfusion (SNMP) on rodent hindlimbs and to validate our protocol in a heterotopic hindlimb transplant model. METHODS: In this optimization study we compared three different solutions during 6 h of SNMP (n = 4 per group). Ten control limbs were stored in a preservation solution on Static Cold Storage [SCS]). During SNMP we monitored arterial flowrate, lactate levels, and edema. After SNMP, muscle biopsies were taken for histology examination, and energy charge analysis. We validated the best perfusion protocol in a heterotopic limb transplantation model with 30-d follow up (n = 13). As controls, we transplanted untreated limbs (n = 5) and hindlimbs preserved with either 6 or 24 h of SCS (n = 4 and n = 5). RESULTS: During SNMP, arterial outflow increased, and lactate clearance decreased in all groups. Total edema was significantly lower in the HBOC-201 group compared to the BSA group (P = 0.005), 4.9 (4.3-6.1) versus 48.8 (39.1-53.2) percentage, but not to the BSA + PEG group (P = 0.19). Energy charge levels of SCS controls decreased 4-fold compared to limbs perfused with acellular oxygen carrier HBOC-201, 0.10 (0.07-0.17) versus 0.46 (0.42-0.49) respectively (P = 0.002). CONCLUSIONS: Six hours ex vivo SNMP of rodent hindlimbs using an acellular oxygen carrier HBOC-201 results in superior tissue preservation compared to conventional SCS.

Local FK506 implants in non-human primates to prevent early acute rejection in vascularized composite allografts.

BACKGROUND: Previous vascularized composite allograft (VCA) studies from our laboratory have shown that topical FK506 delivery in non-human primates (NHPs) was limited by inadequate dermal penetration and rejection persisted. Herein, we report the first utilization of FK506 via subcutaneously implanted discs to mitigate VCA rejection in NHPs. METHODS: Full major histocompatibility complex (MHC)-mismatched NHP pairs underwent partial-face VCA and FK506 disc implantation along the suture line. All allotransplants were maintained post-operatively for two months on the FK506 discs, methylprednisolone, mycophenolate mofetil, and supplemented with intramuscular FK506 if necessary. Group 1 (n=4) was used for optimization of the implant, while Group 2 (n=3) underwent delayed bone marrow transplantation (DBMT) after two months. VCA skin biopsies and peripheral blood samples were obtained for serial assessment of rejection and mixed chimerism by histopathology and flow cytometry respectively. RESULTS: In Group 1, two technical failures occurred. Of the remaining two NHPs, one developed supratherapeutic levels of FK506 (50-120 ng/mL) and had to be euthanized on postoperative day (POD) 12. Reformulation of the implant resulted in stable FK506 levels (20-30 ng/mL) up to POD12 when further intramuscular (IM) FK506 injections were necessitated. In Group 2, two NHPs survived to undergo conditioning and one successfully developed chimerism at 2-3 weeks post-DBMT (96-97% granulocytes and 7-11% lymphocytes of recipient-origin). However, all three NHPs had to be terminated from study at POD64, 77 and 86 due to underlying post-transplant lymphoproliferative disorder. All VCAs remained rejection-free up to study endpoint otherwise. CONCLUSIONS: This study shows preliminary results of local FK506 implants in potentially mitigating VCA acute rejection for tolerance protocols based on mixed chimerism approach.

Deep inferior epigastric perforator free flap in elderly women for breast reconstruction: The experience of a tertiary referral center and a literature review.

BACKGROUND: A general belief is to consider elderly patients as poor candidates for free flap reconstruction, which does not reflect our 20-year experience for breast reconstruction (BR). The aim of this study was to determine the safety and benefits of BR using deep inferior epigastric perforator (DIEP) free flap in the elderly population. METHODS: We conducted a retrospective study of all consecutive BRs using DIEP flaps in patients 65 years or older at the European Georges Pompidou Hospital from January 2011 to December 2019. Postoperative complications were reported as minor or major. We used a descriptive approach to analyze the main characteristics of the patients included. Surgical patient-reported outcomes and quality of life were assessed using the validated BREAST-Q questionnaire. RESULTS: Eighty-three DIEP flaps were performed in 79 patients (4 bilateral flaps) for BR. Sixty-six percent of the patients (52/79) did not present any complication. Total flap loss occurred in 3 BR (3.6%), arterial thrombosis in 4 BR (4.8%), and venous thrombosis in 8 BR (9.6%). The average duration of inpatient stay was 9.5 (±2.7) days. Forty-one of 69 eligible patients completed the questionnaire (response rate 59.4%). Patients reported high satisfaction and well-being scores. The mean Q score for psychosocial well-being was 75.4 (±16.7) and 59 (±13.3) for satisfaction with breasts. CONCLUSION: In our retrospective cohort, DIEP flap BR in elderly population had similar success and complication rates compared with those in younger patients, as well as high satisfaction scores. The free flap should be encouraged for BR in women over 65 years of age, and personal motivation as well as physiological age considered as main criteria for patient selection.

Facial Trauma 8 years after a Face Transplantation.

Over the past 20 years, vascularized composite allografts (VCAs) have emerged as a realistic option in reconstructive surgery. Long-term follow-up reports indicate that face transplant patients have gained in quality of life and social integration. However, they require close monitoring of their immunosuppressive therapy because they are at high-risk for acute rejection episodes, leading eventually to chronic rejection and allograft loss. Reported acute rejection episodes in VCA recipients occur due to low immunosuppressive therapy (mainly due to lack of patient compliance or decreased doses of immunosuppressants to counter side-effects). Repeated mechanical traumas have recently been shown to trigger acute rejection episodes, especially in hand transplant patients. This article reports our experience of a 10-year follow-up of a 57-year-old face transplant patient and the management of his accidental facial trauma. To our knowledge, our patient is the first to undergo a major trauma on his VCA endangering his graft function and vitality. This report discusses the management of an acute surgical situation in those particular patients, and the challenges that arise to avoid acute rejection of the allograft. Ten years into his face transplant and at 18 months follow-up after his facial trauma, our patient shows great aesthetic and functional outcomes and remains rejection-free; a very encouraging result for all VCA candidates.

In Vivo Activity of Genetically Modified Cells Preseeded in Rat Vascularized Composite Allografts.

OBJECTIVE: Transplantation of the hand or face, known as vascularized composite allotransplantation (VCA), has revolutionized reconstructive surgery. Notwithstanding, there are still several areas of improvement to mitigate immune rejection while sparing systemic adverse effects. The goal of this study was to evaluate the engraftment and viability of a genetically modified cell population pre-engrafted into a VCA transplant, to potentially act as a local biosensor to report and modify the graft in vivo. A rat fibroblast cell line genetically modified to secrete Gaussia-Luciferase (gLuc), which served as a constitutive biomarker of cells, was incorporated into a VCA to study the viability of biosensor cells in a syngeneic rat heterotopic partial hindlimb transplantation model. RESULTS: Five perfusions were first performed as engineering runs to have a stable limb perfusion protocol, followed by 3 perfusions to analyze the cell engraftment during machine perfusion, and finally 4 perfusions to study in vivo persistence of the cell biosensors. Blood samples were collected to monitor gLuc secretion during perfusion and postoperatively. A time-dependent increase in gLuc secretion in the limb perfusion outflow during machine perfusion indirectly verified the presence of biosensors within the graft. After the ex vivo perfusion, VCA hindlimbs were analyzed for near infrared fluorescence emission that showed a presence of dyed engineered cells in all areas of the limbs. Postoperatively, gLuc was detectable 4 to 5 days after transplantation (W = 16, P = .02857). This study demonstrated that engineered cells could be successfully preimplanted into VCAs-an important step toward development of an in vivo biosensor platform to use in modulating acute VCA outcomes.

Combating hypoxemia in COVID-19 patients with a natural oxygen carrier, HEMO2Life® (M101).

BACKGROUND: 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. CONSEQUENCES OF THE HYPOTHESIS: The expected benefits of HEMO2Life® for COVID-19 patients are improved survival, avoidance of tracheal intubation, shorter oxygen supplementation, and the possibility of treating a larger number of patients as molecular respirator without to use an invasive machine.

Toward Development of the Delayed Tolerance Induction Protocol for Vascularized Composite Allografts in Nonhuman Primates.

BACKGROUND: Transplantation of vascularized composite allografts is limited mainly by the need for life-long immunosuppression. The consequent side effects and looming specter of chronic rejection portend eventual allograft loss. Development of tolerogenic protocols is thus of utmost importance to the field of vascularized composite allograft transplantation. METHODS: With a modified delayed tolerance induction protocol, 10 cynomolgus macaques received hand (n = 2) or face vascularized composite allografts across both full and haploidentical major histocompatibility complex barriers before donor bone marrow transplantation at a later date. Protocol and for-cause allograft skin biopsies were performed for immunohistochemical analysis and analysis of donor-recipient leukocyte contribution; mixed chimerism in peripheral blood and in vitro immune responses were assessed serially. RESULTS: Before bone marrow transplantation, maintenance immunosuppression for 4 months led to lethal complications, including posttransplant lymphoproliferative disorder (in two of four recipients), which necessitated early study termination. Shortening the maintenance period to 2 months was clinically relevant and allowed all subsequent subjects (n = 6) to complete the delayed tolerance induction protocol. Acute rejection developed within the first 2 to 4 weeks after transplantation, with corresponding near-complete turnover of allograft leukocytes from donor to recipient origin, but donor-specific antibodies remained negative. After bone marrow transplantation, mixed chimerism failed to develop, although carboxyfluorescein succinimidyl ester mixed lymphocyte reaction demonstrated generalized unresponsiveness. However, the accrual of subsequent rejection episodes eventually culminated in graft vasculopathy and irreversible allograft loss. CONCLUSIONS: Despite the various advantages of the delayed tolerance induction protocol, it failed to reliably induce mixed chimerism and thus immunologic tolerance to vascularized composite allografts, given currently available immunosuppression treatment options. Ongoing work shows promise in overcoming these limitations.

Reconstruction of post-burn anterior neck contractures using a butterfly design free anterolateral thigh perforator flap.

Anterior neck burns represent a major reconstructive challenge due to severe sequalae including restriction in movement and poor aesthetic outcomes. Common treatment options include skin grafting with/without dermal matrices, and loco-regional and distant free flap transfers with/without prior tissue expansion. Such variation in technique is largely influenced by the extent of burn injury requiring resurfacing. In order to optimize like-for-like reconstruction of the anterior neck, use of wide, thin and long flaps such as the anterolateral thigh (ALT) perforator flap have been reported with promising results. Of note, some patients have a tendency towards severe scar contractures, which may be contributed by the greater extent of inflammation during wound healing. We report our experience at 4 years' follow-up after secondary reconstruction of severe, anterior neck burn contractures in two patients by harvesting the ALT flap with a butterfly design. This technique provides adequate wound resurfacing of the burned neck and surrounding areas, and provides good neck extensibility by addressing both anterior and lateral aspects of the scar defect simultaneously. Such a flap design reduces tension on wound edges and thus, the risk of contracture recurrence in what remains a particularly challenging type of burn reconstruction.

Graft vasculopathy of vascularized composite allografts in humans: a literature review and retrospective study.

Mechanisms of chronic rejection of vascularized composite allografts (VCA) remain poorly understood and likely present along a spectrum of highly varied clinicopathological findings. Across both animal and human VCA however, graft vasculopathy (GV) has been the most consistent pathological finding resulting clinically in irreversible allograft dysfunction and eventual loss. A literature review of all reported clinical VCA cases with documented GV up to December 2018 was thus performed to elucidate the possible mechanisms involved. Relevant data extracted include C4d deposition, donor-specific antibody (DSA) formation, extent of human leukocyte antigen (HLA) mismatch, pretransplant panel reactive antibody levels, induction and maintenance immunosuppression used, the number of preceding acute rejection episodes, and time to histological confirmation of GV. Approximately 6% (13 of 205) of all VCA patients reported to date developed GV at a mean of 6 years post-transplantation. 46% of these patients have either lost or had their VCAs removed. Neither C4d nor DSA alone was predictive of GV development; however, when both are present, VCA loss appears inevitable due to progressive GV. Of utmost concern, GV in VCA does not appear to be abrogated by currently available immunosuppressive treatment and is essentially irreversible by the time of diagnosis with allograft loss a likely eventuality.