Gal - T Knockout Porcine Nerve Xenografts Support Axonal Regeneration in a Rodent Sciatic Nerve Model.

BACKGROUND: Nerve xenografts harvested from transgenic α1,3-galactosyltransferase knockout (GalT-KO) pigs lack the epitope responsible for hyperacute rejection in pig-to-primate transplants. It is unknown whether these cold preserved nerve grafts support axonal regeneration in another species during and after immunosuppression. In this study, we compare outcomes between autografts and cold preserved xenografts in a rat sciatic model of nerve gap repair. METHODS: Fifty male Lewis rats had a 1 cm sciatic nerve defect repaired using either: autograft and suture (n=10); 1-week or 4-week cold preserved xenograft and suture (n=10 per group); 1-week or 4-week cold preserved xenograft and photochemical tissue bonding using a human amnion wrap (PTB/HAM) (n=10 per group). Rats with xenografts were given tacrolimus until 4 months post-operatively. At 4 and 7 months, rats were euthanized and nerve sections harvested. Monthly sciatic functional index (SFI) scores were calculated. RESULTS: All groups showed increases in SFI scores by 4 and 7 months. The autograft suture group had the highest axon density at 4 and 7 months. The largest decrease in axon density from 4 to 7 months was in the 1-week cold preserved PTB/HAM group. The only significant difference between group SFI scores occurred at 5 months, when both 1-week cold preserved groups had significantly lower scores than the 4-week cold preserved suture group. CONCLUSIONS: Our results in the rat sciatic model suggest that GalT-KO nerve xenografts may be viable alternatives to autografts and demonstrate the need for further studies of long-gap repair and comparison with acellular nerve allografts. CLINICAL RELEVANCE: This proof-of-concept study in the rat sciatic model demonstrates that cold preserved GalT-KO porcine xenografts support axonal regeneration, as well as axonal viability following immunosuppression withdrawal. These results further suggest a role for both cold preservation and photochemical tissue bonding in modulating the immunological response at the nerve repair site.

The efficacy of vitamin E in preventing arthrofibrosis after joint replacement.

BACKGROUND: Arthrofibrosis is a joint disorder characterized by excessive scar formation in the joint tissues. Vitamin E is an antioxidant with potential anti-fibroblastic effect. The aim of this study was to establish an arthrofibrosis rat model after joint replacement and assess the effects of vitamin E supplementation on joint fibrosis. METHODS: We simulated knee replacement in 16 male Sprague-Dawley rats. We immobilized the surgical leg with a suture in full flexion. The control groups were killed at 2 and 12 weeks (n = 5 per group), and the test group was supplemented daily with vitamin E (0.2 mg/mL) in their drinking water for 12 weeks (n = 6). We performed histological staining to investigate the presence and severity of arthrofibrosis. Immunofluorescent staining and α2-macroglobulin (α2M) enzyme-linked immunosorbent assay (ELISA) were used to assess local and systemic inflammation. Static weight bearing (total internal reflection) and range of motion (ROM) were collected for functional assessment. RESULTS: The ROM and weight-bearing symmetry decreased after the procedure and recovered slowly with still significant deficit at the end of the study for both groups. Histological analysis confirmed fibrosis in both lateral and posterior periarticular tissue. Vitamin E supplementation showed a moderate anti-inflammatory effect on the local and systemic levels. The vitamin E group exhibited significant improvement in ROM and weight-bearing symmetry at day 84 compared to the control group. CONCLUSIONS: This model is viable for simulating arthrofibrosis after joint replacement. Vitamin E may benefit postsurgical arthrofibrosis, and further studies are needed for dosing requirements.

A Swine Burn Model for Investigating the Healing Process in Multiple Depth Burn Wounds.

Burn wound healing is a complex and long process. Despite extensive experience, plastic surgeons and specialized teams in burn centers still face significant challenges. Among these challenges, the extent of the burned soft tissue can evolve in the early phase, creating a delicate balance between conservative treatments and necrosing tissue removal. Thermal burns are the most common type, and burn depth varies depending on multiple parameters, such as temperature and exposure time. Burn depth also varies in time, and the secondary aggravation of the "shadow zone" remains a poorly understood phenomenon. In response to these challenges, several innovative treatments have been studied, and more are in the early development phase. Nanoparticles in modern wound dressings and artificial skin are examples of these modern therapies still under evaluation. Taken together, both burn diagnosis and burn treatments need substantial advancements, and research teams need a reliable and relevant model to test new tools and therapies. Among animal models, swine are the most relevant because of their strong similarities in skin structure with humans. More specifically, Yucatan minipigs show interesting features such as melanin pigmentation and slow growth, allowing for studying high phototypes and long-term healing. This article aims to describe a reliable and reproducible protocol to study multi-depth burn wounds in Yucatan minipigs, enabling long-term follow-up and providing a relevant model for diagnosis and therapeutic studies.

Single-cell analysis of immune and stroma cell remodeling in clear cell renal cell carcinoma primary tumors and bone metastatic lesions.

BACKGROUND: Despite therapeutic advances, once a cancer has metastasized to the bone, it represents a highly morbid and lethal disease. One third of patients with advanced clear cell renal cell carcinoma (ccRCC) present with bone metastasis at the time of diagnosis. However, the bone metastatic niche in humans, including the immune and stromal microenvironments, has not been well-defined, hindering progress towards identification of therapeutic targets. METHODS: We collected fresh patient samples and performed single-cell transcriptomic profiling of solid metastatic tissue (Bone Met), liquid bone marrow at the vertebral level of spinal cord compression (Involved), and liquid bone marrow from a different vertebral body distant from the tumor site but within the surgical field (Distal), as well as bone marrow from patients undergoing hip replacement surgery (Benign). In addition, we incorporated single-cell data from primary ccRCC tumors (ccRCC Primary) for comparative analysis. RESULTS: The bone marrow of metastatic patients is immune-suppressive, featuring increased, exhausted CD8 + cytotoxic T cells, T regulatory cells, and tumor-associated macrophages (TAM) with distinct transcriptional states in metastatic lesions. Bone marrow stroma from tumor samples demonstrated a tumor-associated mesenchymal stromal cell population (TA-MSC) that appears to be supportive of epithelial-to mesenchymal transition (EMT), bone remodeling, and a cancer-associated fibroblast (CAFs) phenotype. This stromal subset is associated with poor progression-free and overall survival and also markedly upregulates bone remodeling through the dysregulation of RANK/RANKL/OPG signaling activity in bone cells, ultimately leading to bone resorption. CONCLUSIONS: These results provide a comprehensive analysis of the bone marrow niche in the setting of human metastatic cancer and highlight potential therapeutic targets for both cell populations and communication channels.

Photochemical Tissue Bonding of Amnion Allograft Membranes for Peripheral Nerve Repair: A Biomechanical Analysis.

BACKGROUND: Photochemical tissue bonding (PTB) is a technique for peripheral nerve repair in which a collagenous membrane is bonded around approximated nerve ends. Studies using PTB with cryopreserved human amnion have shown promising results in a rat sciatic nerve transection model including a more rapid and complete return of function, larger axon size, and thicker myelination than suture repair. Commercial collagen membranes, such as dehydrated amnion allograft, are readily available, offer ease of storage, and have no risk of disease transmission or tissue rejection. However, the biomechanical properties of these membranes using PTB are currently unknown in comparison to PTB of cryopreserved human amnion and suture neurorrhaphy. METHODS: Rat sciatic nerves (n = 10 per group) were transected and repaired using either suture neurorrhaphy or PTB with one of the following membranes: cryopreserved human amnion, monolayer human amnion allograft (crosslinked and noncrosslinked), trilayer human amnion/chorion allograft (crosslinked and noncrosslinked), or swine submucosa. Repaired nerves were subjected to mechanical testing. RESULTS: During ultimate stress testing, the repair groups that withstood the greatest strain increases were suture neurorrhaphy (69 ± 14%), PTB with crosslinked trilayer amnion (52 ± 10%), and PTB with cryopreserved human amnion (46 ± 20%), although the differences between these groups were not statistically significant. Neurorrhaphy repairs had a maximum load (0.98 ± 0.30 N) significantly greater than all other repair groups except for noncrosslinked trilayer amnion (0.51 ± 0.27 N). During fatigue testing, all samples repaired with suture, or PTBs with either crosslinked or noncrosslinked trilayer amnion were able to withstand strain increases of at least 50%. CONCLUSION: PTB repairs with commercial noncrosslinked amnion allograft membranes can withstand physiological strain and have comparable performance to repairs with human amnion, which has demonstrated efficacy in vivo. These results indicate the need for further testing of these membranes using in vivo animal model repairs.

Gelatin methacryloyl hydrogel with and without dental pulp stem cells for TMJ regeneration: An in vivo study in rabbits.

BACKGROUND: In the last decade, tissue-engineering strategies for regenerating the temporomandibular joint (TMJ) have been investigated. This may be a promising strategy for the minimally invasive restoration of joint integrity. OBJECTIVES: To evaluate whether dental pulp stem cells (DPSCs) loaded in a light-occured hydrogel made of gelatin methacryloyl (GelMA) enhance the regeneration of osteochondral defects in the rabbit TMJ. MATERIALS AND METHODS: Defects were filled with GelMA alone (control group; n = 4) or filled with GelMA loaded with rabbit DPSCs (experimental group; n = 4), In one group, the TMJ capsule was opened without creating a defect (sham group; n = 2). The following micro-CT parameters were analysed: bone volume to total volume ratio (BV/TV%) and bone mineral density (BMD). Histological evaluation was performed to assess cartilage regeneration features. A semi-quantitative scoring system was also used to evaluate the defects. RESULTS: All groups had no statistical difference regarding the micro-CT parameters. The highest mean healing score was found for the experimental group. After 4 weeks, there were no signs of hydrogel in either group or no signs of inflammation in the adjacent tissues. The tissue formed in the defect was dense fibrous connective tissue. CONCLUSION: Adding DPSCs to GelMA did not provide a regenerative enhancement in TMJ osteochondral defects. This resulted in similar micro-CT parameters after 4 weeks of healing, with improved signs of subchondral bone regeneration but no cartilage regeneration.

Towards Optimizing Sub-Normothermic Machine Perfusion in Fasciocutaneous Flaps: 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.

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

Autonomization is a physiological process allowing a flap to develop neo-vascularization 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.

Adult Prg4+ progenitors repair long-term articular cartilage wounds in vivo.

The identity and origin of the stem/progenitor cells for adult joint cartilage repair remain unknown, impeding therapeutic development. Simulating the common therapeutic modality for cartilage repair in humans, i.e., full-thickness microfracture joint surgery, we combined the mouse full-thickness injury model with lineage tracing and identified a distinct skeletal progenitor cell type enabling long-term (beyond 7 days after injury) articular cartilage repair in vivo. Deriving from a population with active Prg4 expression in adulthood while lacking aggrecan expression, these progenitors proliferate, differentiate to express aggrecan and type II collagen, and predominate in long-term articular cartilage wounds, where they represent the principal repair progenitors in situ under native repair conditions without cellular transplantation. They originate outside the adult bone marrow or superficial zone articular cartilage. These findings have implications for skeletal biology and regenerative medicine for joint injury repair.

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.

Bone Marrow Stem Cells with Tissue-Engineered Scaffolds for Large Bone Segmental Defects: A Systematic Review.

Critical-sized bone defects (CSBDs) represent a significant clinical challenge, stimulating researchers to seek new methods for successful bone reconstruction. The aim of this systematic review is to assess whether bone marrow stem cells (BMSCs) combined with tissue-engineered scaffolds have demonstrated improved bone regeneration in the treatment of CSBD in large preclinical animal models. A search of electronic databases (PubMed, Embase, Web of Science, and Cochrane Library) focused on in vivo large animal studies identified 10 articles according to the following inclusion criteria: (1) in vivo large animal models with segmental bone defects; (2) treatment with tissue-engineered scaffolds combined with BMSCs; (3) the presence of a control group; and (4) a minimum of a histological analysis outcome. Animal research: reporting of in Vivo Experiments guidelines were used for quality assessment, and Systematic Review Center for Laboratory animal Experimentation's risk of bias tool was used to define internal validity. The results demonstrated that tissue-engineered scaffolds, either from autografts or allografts, when combined with BMSCs provide improved bone mineralization and bone formation, including a critical role in the remodeling phase of bone healing. BMSC-seeded scaffolds showed improved biomechanical properties and microarchitecture properties of the regenerated bone when compared with untreated and scaffold-alone groups. This review highlights the efficacy of tissue engineering strategies for the repair of extensive bone defects in preclinical large-animal models. In particular, the use of mesenchymal stem cells, combined with bioscaffolds, seems to be a successful method in comparison to cell-free scaffolds.

Continuous versus Pulsatile Flow in 24-Hour Vascularized Composite Allograft Machine Perfusion in Swine: A Pilot Study.

INTRODUCTION: Multiple perfusion systems have been investigated on vascularized composite allografts, with various temperatures and different preservation solutions, most using continuous flow (CF). However, physiological flow is pulsatile and provides better outcomes in kidney and lung ex vivo perfusions. The objective of this pilot study is to compare pulsatile flow (PF) with CF in our 24-h subnormothermic machine perfusion protocol for swine hindlimbs. METHODS: Partial hindlimbs were harvested from Yorkshire pigs and perfused with a modified Steen solution at 21°C for 24 h either with CF (n = 3) or with pulsatile flow (PF) at 60 beats/min (n = 3). Perfusion parameters, endothelial markers, and muscle biopsies were assessed at different timepoints. RESULTS: Overall, lactate levels were significantly lower in the PF group (P = 0.001). Glucose uptake and potassium concentration were similar in both groups throughout perfusion. Total nitric oxide levels were significantly higher in the PF group throughout perfusion (P = 0.032). Nitric oxide/endothelin-1 ratio also tends to be higher in the PF group, reflecting a potentially better vasoconductivity with PF, although not reaching statistical significance (P = 0.095). Arterial resistances were higher in the PF group (P < 0.001). Histological assessment did not show significant difference in muscular injury between the two groups. Weight increased quicker in the CF group but reached similar values with the PF after 24 h. CONCLUSIONS: This pilot study suggests that PF may provide superior preservation of vascularized composite allografts when perfused for 24 h at subnormothermic temperatures, with potential improvement in endothelial function and decreased ischemic injury.

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.

Light-Activated Vascular Anastomosis.

Background. There have been few advances in technique since vascular anastomosis was performed with silk suture on a curved needle in 1902. This technique results in disruption of the endothelium with exposed intraluminal suture, both of which may lead to thrombocyte aggregation, intimal hyperplasia, and vascular stenosis. A variety of alternative techniques have been explored, with limited success. Photochemical tissue bonding (PTB) is a light-activated methodology of rapidly cross-linking tissue interfaces at the molecular level. Herein, we describe a new technique for anastomosis of venous interposition graft in an ovine model of femoral artery bypass utilizing PTB. Methods. Polypay specific pathogen free sheep (n = 5; 40-45 kg) underwent femoral artery bypass utilizing saphenous vein. The femoral artery was transected and reversed saphenous vein was implanted as an interposition graft. The proximal anastomosis was created as a vein-over-artery cuff utilizing PTB, and the distal anastomosis was created with standard interrupted 8-0 polypropylene suture. Four weeks post-index operation, femoral angiogram was performed to evaluate patency, tortuosity, and luminal diameter. All bypass grafts were harvested and longitudinal and transverse histological sections from the proximal anastomosis were analyzed. Results. The PTB anastomoses (n = 5) were immediately watertight and patent. All animals survived the 28-day study duration. Angiography revealed patent grafts with no aneurysm or stenosis (n = 5). Histologic examination revealed integration of the venous endothelium with the arterial adventitia. Conclusion. Photochemical tissue bonding creates an immediate strong, watertight vascular anastomosis that can withstand physiologic arterial pressure and remains patent at 28 days without the need for intraluminal suture.

Exceeding the Limits of Static Cold Storage in Limb Transplantation Using Subnormothermic Machine Perfusion.

BACKGROUND: For 50 years, static cold storage (SCS) has been the gold standard for solid organ preservation in transplantation. Although logistically convenient, this preservation method presents important constraints in terms of duration and cold ischemia-induced lesions. We aimed to develop a machine perfusion (MP) protocol for recovery of vascularized composite allografts (VCA) after static cold preservation and determine its effects in a rat limb transplantation model. METHODS: Partial hindlimbs were procured from Lewis rats and subjected to SCS in Histidine-Tryptophan-Ketoglutarate solution for 0, 12, 18, 24, and 48 hours. They were then either transplanted (Txp), subjected to subnormothermic machine perfusion (SNMP) for 3 hours with a modified Steen solution, or to SNMP + Txp. Perfusion parameters were assessed for blood gas and electrolytes measurement, and flow rate and arterial pressures were monitored continuously. Histology was assessed at the end of perfusion. For select SCS durations, graft survival and clinical outcomes after transplantation were compared between groups at 21 days. RESULTS: Transplantation of limbs preserved for 0, 12, 18, and 24-hour SCS resulted in similar survival rates at postoperative day 21. Grafts cold-stored for 48 hours presented delayed graft failure (p = 0.0032). SNMP of limbs after 12-hour SCS recovered the vascular resistance, potassium, and lactate levels to values similar to limbs that were not subjected to SCS. However, 18-hour SCS grafts developed significant edema during SNMP recovery. Transplantation of grafts that had undergone a mixed preservation method (12-hour SCS + SNMP + Txp) resulted in better clinical outcomes based on skin clinical scores at day 21 post-transplantation when compared to the SCS + Txp group (p = 0.01613). CONCLUSION: To date, VCA MP is still limited to animal models and no protocols are yet developed for graft recovery. Our study suggests that ex vivo SNMP could help increase the preservation duration and limit cold ischemia-induced injury in VCA transplantation.

Photo-crosslinked GelMA loaded with dental pulp stem cells and VEGF to repair critical-sized soft tissue defects in rats.

BACKGROUND: Tissue engineering of skin and mucosa is essential for the esthetic and functional reconstruction of individuals disfigured by trauma, resection surgery, or severe burns while overcoming the limited amount of autograft and donor site morbidity. PURPOSE: We aimed to determine whether a combination of Gelatin-methacryloyl (GelMA) hydrogel scaffold alone or loaded with either dental pulp stem cells (DPSCs) and/or vascular endothelial growth factor (VEGF) could improve skin wound healing in rats. MATERIALS AND METHODS: Four 10 mm full-thickness skin defects were created on the dorsum of 15 Sprague-Dawley rats. The wounds were treated with GelMA alone, GelMA+DPSCs, or GelMA+DPSCs+VEGF. Unprotected wounds were used as controls. Animals were euthanized at 1-, 2-, and 4 weeks post-surgery, and the healing wounds were harvested for clinical, histological, and RT-PCR analysis. RESULTS: No signs of clinical inflammation were observed among all groups. Few and sparse mononuclear inflammatory cells were observed in GelMA+DPSCs and GelMA+DPSCs+VEGF groups at 2 weeks, with complete epithelialization of the wounds. At 4 weeks, the epidermis in GelMA+DPSCs and GelMA+DPSCs+VEGF groups was indistinguishable from the empty defect and GelMA groups. The decrease in cellularity and increase in density of collagen fibers were observed over time in both GelMA+DPSCs and GelMA+DPSCs+VEGF groups but were more evident in the GelMA+DPSCs+VEGF group. The GelMA+DPSCs+VEGF group showed a higher expression of the KER 10 gene at all time points compared with the other groups. Expression of Col1 A1 and TGFß-1 were not statistically different over time neither among the groups. CONCLUSION: GelMA scaffolds loaded with DPSCs, and VEGF accelerated the re-epithelialization of skin wounds.

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.

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, failed by the first steps of the reconstructive ladder. Despite promising aesthetic and functional outcomes, the main limiting factor remains the need for a drastically applied lifelong immunosuppression and its well-known medical risks, preventing broader indications. Therefore, lifting the immune barrier in VCA is essential to tip the ethical scale and improve patients' quality of life using the most advanced surgical techniques. De novo creation of a patient-specific graft is the upcoming breakthrough in reconstructive transplantation. Using tissue engineering techniques, VCAs can be freed of donor cells and customized for the recipient through perfusion-decellularization-recellularization. To develop these new technologies, a large-scale animal VCA model is necessary. Hence, swine fascio-cutaneous flaps, composed of skin, fat, fascia, and vessels, represent an ideal model for preliminary studies in VCA. Nevertheless, most VCA models described in the literature include muscle and bone. This work reports a reliable and reproducible technique for saphenous fascio-cutaneous flap harvest in swine, a practical tool for various research fields, especially vascularized composite tissue engineering.