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Current methods of storing explanted donor livers at 4 °C in University of Wisconsin (UW) solution result in loss of graft function and ultimately lead to less-than-ideal outcomes post transplantation. Our lab has previously shown that supplementing UW solution with 35-kilodalton polyethylene glycol (PEG) has membrane stabilizing effects for cold stored primary rat hepatocytes in suspension. Expanding on past studies, we here investigate if PEG has the same beneficial effects in an adherent primary rat hepatocyte cold storage model. In addition, we investigated the extent of cold-induced apoptosis through treating cold-stored hepatocytes with pan caspase inhibitor emricasan. In parallel to storage at the current cold storage standard of 4 °C, we investigated the effects of lowering the storage temperature to -4 °C, at which the storage solution remains ice-free due to the supercooling phenomenon. We show the addition of 5 % PEG to the storage medium significantly reduced the release of lactate dehydrogenase (LDH) in plated rat hepatocytes and a combinatorial treatment with emricasan maintains hepatocyte viability and morphology following recovery from cold storage. These results show that cold-stored hepatocytes undergo multiple mechanisms of cold-induced injury and that PEG and emricasan treatment in combination with supercooling may improve cell and organ preservation.
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Apoptose , Inibidores de Caspase , Criopreservação , Hepatócitos , L-Lactato Desidrogenase , Soluções para Preservação de Órgãos , Polietilenoglicóis , Animais , Hepatócitos/efeitos dos fármacos , Polietilenoglicóis/farmacologia , Ratos , Soluções para Preservação de Órgãos/farmacologia , Criopreservação/métodos , Masculino , L-Lactato Desidrogenase/metabolismo , Apoptose/efeitos dos fármacos , Inibidores de Caspase/farmacologia , Rafinose/farmacologia , Células Cultivadas , Alopurinol/farmacologia , Crioprotetores/farmacologia , Temperatura Baixa , Glutationa/metabolismo , Glutationa/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Insulina/farmacologia , Adenosina/farmacologia , Preservação de Órgãos/métodos , Ratos Sprague-Dawley , Ácidos PentanoicosRESUMO
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.
Assuntos
Aloenxertos Compostos , Preservação de Órgãos , Suínos , Animais , Projetos Piloto , Preservação de Órgãos/métodos , Fluxo Pulsátil/fisiologia , Óxido Nítrico , Perfusão/métodosRESUMO
Liver tissue engineering aims to create transplantable liver grafts that can serve as substitutes for donor's livers. One major challenge in creating a fully functional liver tissue has been to recreate the biliary drainage in an engineered liver construct through integration of bile canaliculi (BC) with the biliary ductular network that would enable the clearance of bile from the hepatocytes to the host duodenum. In this study, we show the formation of such a hepatic microtissue by coculturing rat primary hepatocytes with cholangiocytes and stromal cells. Our results indicate that within the spheroids, hepatocytes maintained viability and function for up to 7 days. Viable hepatocytes became polarized by forming BC with the presence of tight junctions. Morphologically, hepatocytes formed the core of the spheroids, while cholangiocytes resided at the periphery forming a monolayer microcysts and tubular structures extending outward. The spheroids were subsequently cultured in clusters to create a higher order ductular network resembling hepatic lobule. The cholangiocytes formed functional biliary ductular channels in between hepatic spheroids that were able to collect, transport, and secrete bile. Our results constitute the first step to recreate hepatic building blocks with biliary drainage for repopulating the whole liver scaffolds to be used as transplantable liver grafts.
Assuntos
Ductos Biliares/metabolismo , Hepatócitos/metabolismo , Esferoides Celulares/metabolismo , Engenharia Tecidual , Animais , Ductos Biliares/citologia , Células Cultivadas , Hepatócitos/citologia , Fígado , Ratos , Esferoides Celulares/citologiaRESUMO
Tissue engineering scaffolds are intended to provide mechanical and biological support for cells to migrate, engraft and ultimately regenerate the tissue. Development of scaffolds with sustained delivery of growth factors and chemokines would enhance the therapeutic benefits, especially in wound healing. In this study, we incorporated our previously designed therapeutic particles, composed of fusion of elastin-like peptides (ELPs) as the drug delivery platform to keratinocyte growth factor (KGF), into a tissue scaffold, alloderm. The results demonstrated that sustained KGF-ELP release was achieved and the bioactivity of the released therapeutic particles was shown via cell proliferation assay, as well as a mouse pouch model in vivo, where higher cellular infiltration and vascularization were observed in scaffolds functionalized with KGF-ELPs.
Assuntos
Biopolímeros/química , Colágeno/química , Elastina/química , Alicerces Teciduais/química , Animais , Biopolímeros/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Colágeno/farmacologia , Fator 7 de Crescimento de Fibroblastos/metabolismo , Humanos , Camundongos , Neovascularização Fisiológica/efeitos dos fármacos , Engenharia TecidualRESUMO
A summary of the First Signature Series Event, "Advancements in Cellular Therapies and Regenerative Medicine for Digestive Diseases," held on May 3, 2017, in London, United Kingdom, is presented. Twelve speakers from three continents covered major topics in the areas of cellular therapy and regenerative medicine applied to liver and gastrointestinal medicine as well as to diabetes mellitus. Highlights from their presentations, together with an overview of the global impact of digestive diseases and a proposal for a shared online collection and data-monitoring platform tool, are included in this proceedings. Although growing evidence demonstrate the feasibility and safety of exploiting cell-based technologies for the treatment of digestive diseases, regulatory and methodological obstacles will need to be overcome before the successful implementation in the clinic of these novel attractive therapeutic strategies.
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Terapia Baseada em Transplante de Células e Tecidos/métodos , Gastroenteropatias/terapia , Medicina Regenerativa/métodos , Terapia Baseada em Transplante de Células e Tecidos/tendências , Diabetes Mellitus/patologia , Diabetes Mellitus/terapia , Gastroenteropatias/patologia , Humanos , Hepatopatias/patologia , Hepatopatias/terapia , Medicina Regenerativa/tendênciasRESUMO
Viruses lack the basic machinery needed to replicate and therefore must hijack the host's metabolism to propagate. Virus-induced metabolic changes have yet to be systematically studied in the context of host transcriptional regulation, and such studies shoul offer insight into host-pathogen metabolic interplay. In this work we identified hepatitis C virus (HCV)-responsive regulators by coupling system-wide metabolic-flux analysis with targeted perturbation of nuclear receptors in primary human hepatocytes. We found HCV-induced upregulation of glycolysis, ketogenesis and drug metabolism, with glycolysis controlled by activation of HNF4α, ketogenesis by PPARα and FXR, and drug metabolism by PXR. Pharmaceutical inhibition of HNF4α reversed HCV-induced glycolysis, blocking viral replication while increasing apoptosis in infected cells showing virus-induced dependence on glycolysis. In contrast, pharmaceutical inhibition of PPARα or FXR reversed HCV-induced ketogenesis but increased viral replication, demonstrating a novel host antiviral response. Our results show that virus-induced changes to a host's metabolism can be detrimental to its life cycle, thus revealing a biologically complex relationship between virus and host.
Assuntos
Hepacivirus/metabolismo , Hepatite C/metabolismo , Hepatite C/virologia , Interações Hospedeiro-Patógeno , Receptores Citoplasmáticos e Nucleares/metabolismo , Glicólise , Hepacivirus/efeitos dos fármacos , Hepacivirus/crescimento & desenvolvimento , Hepatócitos/metabolismo , Hepatócitos/virologia , HumanosAssuntos
Aloenxertos Compostos , Animais , Suínos , Projetos Piloto , Fluxo Pulsátil/fisiologia , Perfusão , Transplante HomólogoRESUMO
Treatment for end-stage liver failure is restricted by the critical shortage of donor organs; about 4000 people die in the USA while waiting for a transplantable organ. This situation has been a major driving force behind the rise of tissue engineering to build artificial tissues/organs. Recent advancements in creating transplantable liver grafts using decellularized liver scaffolds bring the field closer to clinical translation. However, a source of readily available and highly functional adult hepatocytes in adequate numbers for regenerative liver therapies still remains unclear. Here, we describe a new method to utilize discarded livers to make transplantable new liver grafts. We show that marginal donor livers damaged due to warm ischemia could be treated with machine perfusion to yield 39 million viable hepatocytes per gram of liver, similar to fresh livers, and these cells could be used to repopulate decellularized liver matrix (DLM) scaffolds to make transplantable liver grafts. The hepatocytes from recovered livers sustained their characteristic epithelial morphology while they exhibited slightly lower protein synthesis functions both in plate cultures and in recellularized liver grafts. The dampened protein synthesis was attributed to residual endoplasmic reticulum stress found in recovered cells. The results here represent a unique approach to reengineer transplantable liver grafts solely from discarded organs.
Assuntos
Hepatócitos/citologia , Regeneração Hepática , Fígado/fisiologia , Engenharia Tecidual/métodos , Animais , Separação Celular , Células Cultivadas , Matriz Extracelular/química , Fígado/química , Fígado/citologia , Perfusão , Ratos , Alicerces Teciduais/químicaRESUMO
PURPOSE OF REVIEW: Organ donation in the United States registered 9079 deceased organ donors in 2015. This high percentage of donations allowed organ transplantation in 29â851 recipients. Despite increasing numbers of transplants performed in comparison with previous years, the numbers of patients that are in need for a transplant increase every year at a higher rate. This reveals that the discrepancy between the demand and availability of organs remains fundamental problem in organ transplantation. RECENT FINDINGS: Development of bioengineered organs represents a promising approach to increase the pool of organs for transplantation. The technology involves obtaining complex three-dimensional scaffolds that support cellular activity and functional remodeling though tissue recellularization protocols using progenitor cells. This innovative approach integrates cross-thematic approaches from specific areas of transplant immunology, tissue engineering and stem cell biology, to potentially manufacture an unlimited source of donor organs for transplantation. SUMMARY: Although bioengineered organs are thought to escape immune recognition, the potential immune reactivity toward each of its components has not been studied in detail. Here, we summarize the host immune response toward different progenitor cells and discuss the potential implications of using nonself biological scaffolds to develop bioengineered organs.
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Doadores de Tecidos , Engenharia Tecidual/métodos , Obtenção de Tecidos e Órgãos/métodos , Morte , HumanosRESUMO
Cutaneous burns are often exacerbated by poor perfusion and subsequent necrosis of the microvasculature surrounding the primary injury. Preservation of these vessels can reduce necrotic tissue expansion and increase success rates of skin graft procedures. Recent work has identified a peptide derived from erythropoietin, ARA290, with the ability to mediate tissue protection in a variety of cell types. Here we demonstrate the advantages of fusing ARA290 to an elastin-like polypeptide (ELP) to salvage microvascular endothelial cells in harsh proteolytic conditions following thermal shock. These fusion proteins were expressed recombinantly in bacterial hosts and rapidly purified by inverse transition cycling. They were shown to spontaneously aggregate into particles at subphysiological temperatures. The bifunctional submicron particles were resistant to digestion in enzymes upregulated after burn injury. Furthermore, the data strongly suggest these ARA290-functionalized particles were superior to treatment with the peptide alone in preventing microvascular cell death in these conditions. The results bring to light an efficient and cost-effective strategy for the delivery therapeutic peptides to proteolytically active wound sites.
Assuntos
Biopolímeros/farmacologia , Elastina/química , Oligopeptídeos/química , Proteólise/efeitos dos fármacos , Proteínas Recombinantes de Fusão/farmacologia , Biopolímeros/química , Biopolímeros/genética , Queimaduras/prevenção & controle , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Elastina/genética , Elastina/farmacologia , Eritropoetina/química , Escherichia coli/genética , Temperatura Alta , Humanos , Microvasos/efeitos dos fármacos , Microvasos/metabolismo , Microvasos/patologia , Oligopeptídeos/genética , Oligopeptídeos/farmacologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Pele/irrigação sanguínea , Cicatrização/efeitos dos fármacosRESUMO
Decellularization of discarded whole livers and their recellularization with patient-specific induced pluripotent stem cells (iPSCs) to develop a functional organ is a promising approach to increasing the donor pool. The effect of extracellular matrix (ECM) of marginal livers on iPSC-hepatocyte differentiation and function has not been shown. To test the effect of donor liver ECM age and steatosis, young and old, as well as no, low, and high steatosis livers, are decellularized. All livers are decellularized successfully. High steatosis livers have fat remaining on the ECM after decellularization. Old donor liver ECM induces lower marker expression in early differentiation stages, compared to young liver ECM, while this difference is closed at later stages and do not affect iPSC-hepatocyte function significantly. High steatosis levels of liver ECM lead to higher albumin mRNA expression and secretion while at later stages of differentiation expression of major cytochrome (CYP) 450 enzymes is highest in low steatosis liver ECM. Both primary human hepatocytes and iPSC-hepatocytes show an increase in fat metabolism marker expression with increasing steatosis levels most likely induced by excess fat remaining on the ECM. Overall, removal of excess fat from liver ECM may be needed for inducing proper hepatic function after recellularization.
Assuntos
Diferenciação Celular , Matriz Extracelular , Fígado Gorduroso , Hepatócitos , Células-Tronco Pluripotentes Induzidas , Fígado , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Matriz Extracelular/metabolismo , Hepatócitos/metabolismo , Hepatócitos/citologia , Fígado/metabolismo , Fígado/patologia , Adulto , Doadores de Tecidos , Pessoa de Meia-Idade , Células Cultivadas , Fatores Etários , Alicerces Teciduais/químicaRESUMO
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.
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Background and Objective: 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. Methods: A literature review was conducted between April and June 2023 on PubMed and Google Scholar Databases. All English and French articles related to bioengineering applied to the field of breast reconstruction were included. We used the Evidence-Based Veterinary Medicine Association (EBVM) Toolkit 14 checklist for narrative reviews as a quality assurance measure and the Scale for the Assessment of Narrative Review Articles (SANRA) tool to self-assess our methodology. Key Content and Findings: Over 130 references related to breast bioengineering were included. The analysis revealed four key applications: enhancing the quality of the skin envelope, improving the viability of fat grafting, creating breast shape and volume via bio-printing, and optimizing nipple reconstruction through engineering techniques. The primary identified approaches revolved around establishing structural support and enhancing cellular viability. Structural techniques predominantly involved the implementation of 3D printed, decellularized, or biocompatible material scaffolds. Meanwhile, promoting cellular content trophicity primarily focused on harnessing the regenerative potential of adipose-derived stem cells (ADSCs) and increasing the tissue's survivability and cell trophicity. Conclusions: Tissue and bioengineering hold immense promise in the field of breast reconstruction, offering a diverse array of approaches. By combining existing techniques with novel advancements, they have the potential to significantly enhance the therapeutic options available to plastic and reconstructive surgeons.
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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.
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Pele , Cicatrização , Suínos , Animais , Humanos , Porco Miniatura , Cicatrização/fisiologia , Bandagens , Modelos Animais de DoençasRESUMO
BACKGROUND & AIMS: Alterations in mitochondrial morphology and function and increased oxidative stresses in hepatocytes are well established in nonalcoholic fatty liver disease (NAFLD). Patients can undergo lifestyle changes, especially in earlier NAFLD stages, to reverse disease-induced phenotypes on a gross level. Yet, little is known about whether mitochondrial function and injuries recover upon reversal. Thus, we elucidated this question and interplays between the cytoskeletal network and mitochondria in the development and reversal of steatosis. METHODS: We cultured primary human hepatocytes stably for 2 weeks and used free fatty acid supplementation to induce steatosis over 7 days and reversed steatosis by free fatty acid withdrawal over the next 7 days. We assessed cytoskeletal and mitochondrial morphologies using immunocytochemistry and confocal microscopy. We evaluated mitochondrial respiration and function via the Seahorse analyzer, in which we fully optimized reagent dosing specifically for human hepatocytes. RESULTS: During early steatosis, intracellular lipid droplets displaced microtubules altering mitochondrial distribution, and disrupted the F-actin network, leading to loss of bile canaliculi in steatotic hepatocytes. Basal mitochondrial respiration, maximum respiratory capacity, and resistance to H2O2-induced cell death also increased as an adaptative response. Upon reversal of steatosis, F-actin and bile canaliculi were restored in hepatocytes. Nevertheless, we observed an increase in elongated mitochondrial branches accompanied by decreases in α-tubulin expression, mitochondrial proton leak, and susceptibility to H2O2-induced cell death. CONCLUSIONS: Despite the restoration of cytoskeletons morphologically upon reversal of steatosis, the mitochondria in hepatocytes were impaired owing to early adaptative respiratory increase. Hepatocytes thus were highly predisposed to H2O2-induced cell death. These results indicate the persistence of potential health risks for recovering NAFLD patients.
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Hepatopatia Gordurosa não Alcoólica , Humanos , Hepatopatia Gordurosa não Alcoólica/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Actinas/metabolismo , Peróxido de Hidrogênio/metabolismo , Hepatócitos/metabolismo , Mitocôndrias/metabolismo , Citoesqueleto/metabolismo , Microtúbulos/metabolismoRESUMO
Current methods of storing explanted donor livers at 4°C in University of Wisconsin (UW) solution result in loss of graft function and ultimately leads to less-than-ideal outcomes post transplantation. Our lab has previously shown that supplementing UW solution with 35-kilodalton polyethylene glycol (PEG) has membrane stabilizing effects for cold stored primary rat hepatocytes in suspension. Expanding on past studies, we here investigate if PEG has the same beneficial effects in an adherent primary rat hepatocyte cold storage model. In addition, we investigated the extent of cold-induced apoptosis through treating cold-stored hepatocytes with pan caspase inhibitor emricasan. In parallel to storage at the current cold storage standard of 4°C, we investigated the effects of lowering the storage temperature to -4°C, at which the storage solution remains ice-free due to the supercooling phenomenon. We show the addition of 5% PEG to the storage medium significantly reduced the release of lactate dehydrogenase (LDH) in plated rat hepatocytes and a combinatorial treatment with emricasan maintains hepatocyte viability and morphology following recovery from cold storage. These results show that cold-stored hepatocytes undergo multiple mechanisms of cold-induced injury and that PEG and emricasan treatment in combination with supercooling may improve cell and organ preservation.
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This review aims to evaluate the current preclinical state of liver bioengineering, the clinical context for liver cell therapies, the cell sources, the delivery routes, and the results of clinical trials for end-stage liver disease. Different clinical settings, such as inborn errors of metabolism, acute liver failure, chronic liver disease, liver cirrhosis, and acute-on-chronic liver failure, as well as multiple cellular sources were analyzed; namely, hepatocytes, hepatic progenitor cells, biliary tree stem/progenitor cells, mesenchymal stromal cells, and macrophages. The highly heterogeneous clinical scenario of liver disease and the availability of multiple cellular sources endowed with different biological properties make this a multidisciplinary translational research challenge. Data on each individual liver disease and more accurate endpoints are urgently needed, together with a characterization of the regenerative pathways leading to potential therapeutic benefit. Here, we critically review these topics and identify related research needs and perspectives in preclinical and clinical settings.
Assuntos
Hepatopatias , Medicina Regenerativa , Humanos , Medicina Regenerativa/métodos , Transplante de Células-Tronco , Hepatopatias/terapia , Hepatopatias/metabolismo , Fígado/metabolismo , HepatócitosRESUMO
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.
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Coleta de Amostras Sanguíneas , Cauda , Ratos , Masculino , Animais , Cauda/cirurgia , Cauda/irrigação sanguínea , Coleta de Amostras Sanguíneas/métodos , Punções , Animais de Laboratório , Veia Subclávia , Veias JugularesRESUMO
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.
Assuntos
Retalhos de Tecido Biológico , Precondicionamento Isquêmico , Ratos , Animais , Artérias Epigástricas/cirurgia , Reprodutibilidade dos Testes , Precondicionamento Isquêmico/métodosRESUMO
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.