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This study investigates the effects of environmentally-relevant concentrations of fluoxetine (FLX, commercial name: Prozac) on wound healing. Pollution of water systems with pharmaceutical and personal care products, including antidepressants such as FLX and other selective serotonin reuptake inhibitors, is a growing environmental concern. Environmentally-relevant FLX concentrations are known to impact physiological functions and behaviour of aquatic animals, however, the effects of exposure on humans are currently unknown. Using a combination of human skin biopsies and a human keratinocyte cell line, we show that exposure to environmental FLX promotes wound closure. We show dose-dependent increases in wound closure with FLX concentrations from 125 ng/l. Using several -omics and pharmaceutical approaches, we demonstrate that the mechanisms underlying enhanced wound closure are increased cell proliferation and serotonin signalling. Transcriptomic analysis revealed 350 differentially expressed genes after exposure. Downregulated genes were enriched in pathways related to mitochondrial function and metabolism, while upregulated genes were associated with cell proliferation and tissue morphogenesis. Kinase profiling showed altered phosphorylation of kinases linked to the MAPK pathway. Consistent with this, phosphoproteomic analyses identified 235 differentially phosphorylated proteins after exposure, with enriched GO terms related to cell cycle, division, and protein biosynthesis. Treatment of skin biopsies and keratinocytes with ketanserin, a serotonin receptor antagonist, reversed the increase in wound closure observed upon exposure. These findings collectively show that exposure to environmental FLX promotes wound healing through modulating serotonin signalling, gene expression and protein phosphorylation, leading to enhanced cell proliferation. Our results justify a transition from the study of behavioural effects of environmental FLX in aquatic animals to the investigation of effects of exposure on wound healing in aquatic and terrestrial animals, including direct impacts on human health.
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Biomaterials capable of promoting wound healing and preventing infections remain in great demand to address the global unmet need for the treatment of chronic wounds. Phosphate-based glasses (PG) have shown potential as bioresorbable materials capable of inducing tissue regeneration, while being replaced by regenerated tissue and releasing therapeutic species. In this work, phosphate-glass-based fibers (PGF) in the system P2O5-CaO-Na2O added with 1, 2, 4, 6, and 10 mol % of the therapeutic metallic ions (TMI) Ag+, Zn2+, and Fe3+ were manufactured via electrospinning of coacervate gels. Coacervation is a sustainable, cost-effective, water-based method to produce PG. All TMI are effective in promoting wound closure (re-epithelialization) in living human skin ex vivo, where the best-performing system is PGF containing Ag+. In particular, PGF with ≥4 mol % of Ag+ is capable of promoting 84% wound closure over 48 h. These results are confirmed by scratch test migration assays, with the PGF-Ag systems containing ≥6 mol % of Ag+, demonstrating significant wound closure enhancement (up to 72%) after 24 h. The PGF-Ag systems are also the most effective in terms of antibacterial activity against both the Gram-positive Staphylococcus aureus and the Gram-negative Escherichia coli. PGF doped with Zn2+ shows antibacterial activity only against S. aureus in the systems containing Zn2+ ≥ 10 mol %. In addition, PGF doped with Fe3+ rapidly accelerates ex vivo healing in patient chronic wound skin (>30% in 48 h), demonstrating the utility of doped PGF as a potential therapeutic strategy to treat chronic wounds.
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Antibacterianos , Escherichia coli , Vidrio , Fosfatos , Staphylococcus aureus , Cicatrización de Heridas , Humanos , Escherichia coli/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacos , Vidrio/química , Antibacterianos/química , Antibacterianos/farmacología , Cicatrización de Heridas/efectos de los fármacos , Fosfatos/química , Fosfatos/farmacología , Plata/química , Plata/farmacología , Zinc/química , Zinc/farmacología , Iones/química , Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Piel/efectos de los fármacos , Pruebas de Sensibilidad MicrobianaRESUMEN
Our skin is home to a diverse community of commensal microorganisms integral to cutaneous function. However, microbial dysbiosis and barrier perturbation increase the risk of local and systemic infection. Staphylococcus aureus is a particularly problematic bacterial pathogen, with high levels of antimicrobial resistance and direct association with poor healing outcome. Innovative approaches are needed to selectively kill skin pathogens, such as S aureus, without harming the resident microbiota. In this study, we provide important data on the selectivity and efficacy of an S aureus-targeted endolysin (XZ.700) within the complex living skin/wound microbiome. Initial cross-species comparison using Nanopore long-read sequencing identified the translational potential of porcine rather than murine skin for human-relevant microbiome studies. We therefore performed an interventional study in pigs to assess the impact of endolysin administration on the microbiome. XZ.700 selectively inhibited endogenous porcine S aureus in vivo, restoring microbial diversity and promoting multiple aspects of wound repair. Subsequent mechanistic studies confirmed the importance of this microbiome modulation for effective healing in human skin. Taken together, these findings strongly support further development of S aureus-targeted endolysins for future clinical management of skin and wound infections.
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Microbiota , Piel , Staphylococcus aureus , Cicatrización de Heridas , Animales , Staphylococcus aureus/efectos de los fármacos , Cicatrización de Heridas/efectos de los fármacos , Piel/microbiología , Microbiota/efectos de los fármacos , Porcinos , Humanos , Ratones , Endopeptidasas , Modelos Animales de Enfermedad , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/tratamiento farmacológico , FemeninoRESUMEN
Pelvic organ prolapse is a disorder that substantially affects the quality of life of millions of women worldwide. The greatest risk factors for prolapse are increased parity and older age, with the largest group requiring surgical intervention being post-menopausal women over 65. Due to ineffective healing in the elderly, prolapse recurrence rates following surgery remain high. Therefore, there is an urgent need to elucidate the cellular and molecular drivers of poor healing in pelvic floor dysfunction to allow effective management and even prevention. Recent studies have uncovered the importance of Arginase 1 for modulating effective healing in the skin. We thus employed novel in vitro and in vivo vaginal injury models to determine the specific role of Arginase 1 in age-related vaginal repair. Here we show, for the first time, that aged rat vaginal wounds have reduced Arginase 1 expression and delayed healing. Moreover, direct inhibition of Arginase 1 in human vaginal epithelial cells also led to delayed scratch-wound closure. By contrast, activation of Arginase 1 significantly accelerated healing in aged vaginal wounds in vivo, to rates comparable to those in young animals. Collectively, these findings reveal a new and important role for Arginase 1 in mediating effective vaginal repair. Targeting age-related Arginase 1 deficiency is a potential viable therapeutic strategy to promote vaginal healing and reduce recurrence rate after surgical repair of pelvic organ prolapse.
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Arginasa/metabolismo , Prolapso de Órgano Pélvico , Mallas Quirúrgicas , Anciano , Animales , Femenino , Procedimientos Quirúrgicos Ginecológicos , Humanos , Calidad de Vida , RatasRESUMEN
Cellular senescence, once thought an artifact of in vitro culture or passive outcome of aging, has emerged as fundamental to tissue development and function. The senescence mechanism importantly halts cell cycle progression to protect against tumor formation, while transiently present senescent cells produce a complex secretome (or SASP) of inflammatory mediators, proteases, and growth factors that guide developmental remodeling and tissue regeneration. Transiently present senescence is important for skin repair, where it accelerates extracellular matrix formation, limits fibrosis, promotes reepithelialization, and modulates inflammation. Unfortunately, advanced age and diabetes drive pathological accumulation of senescent cells in chronic wounds, which is perpetuated by a proinflammatory SASP, advanced glycation end-products, and oxidative damage. Although the biology of wound senescence remains incompletely understood, drugs that selectively target senescent cells are showing promise in clinical trials for diverse pathological conditions. It may not be long before senescence-targeted therapies will be available for the management, or perhaps even prevention, of chronic wounds.
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Senescencia Celular , Neoplasias , Humanos , Senescencia Celular/fisiología , Envejecimiento/fisiología , Inflamación , Neoplasias/metabolismoRESUMEN
Naked mole-rats (NMRs) (Heterocephalus glaber) are long-lived mammals that possess a natural resistance to cancer and other age-related pathologies, maintaining a healthy life span >30 years. In this study, using immunohistochemical and RNA-sequencing analyses, we compare skin morphology, cellular composition, and global transcriptome signatures between young and aged (aged 3â4 vs. 19â23 years, respectively) NMRs. We show that similar to aging in human skin, aging in NMRs is accompanied by a decrease in epidermal thickness; keratinocyte proliferation; and a decline in the number of Merkel cells, T cells, antigen-presenting cells, and melanocytes. Similar to that in human skin aging, expression levels of dermal collagens are decreased, whereas matrix metalloproteinase 9 and matrix metalloproteinase 11 levels increased in aged versus in young NMR skin. RNA-sequencing analyses reveal that in contrast to human or mouse skin aging, the transcript levels of several longevity-associated (Igfbp3, Igf2bp3, Ing2) and tumor-suppressor (Btg2, Cdkn1a, Cdkn2c, Dnmt3a, Hic1, Socs3, Sfrp1, Sfrp5, Thbs1, Tsc1, Zfp36) genes are increased in aged NMR skin. Overall, these data suggest that specific features in the NMR skin aging transcriptome might contribute to the resistance of NMRs to spontaneous skin carcinogenesis and provide a platform for further investigations of NMRs as a model organism for studying the biology and disease resistance of human skin.
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Proteínas Inmediatas-Precoces , Envejecimiento de la Piel , Animales , Humanos , Ratones , Genes Supresores de Tumor , Proteínas de Homeodominio/genética , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Longevidad/genética , Metaloproteinasa 11 de la Matriz/genética , Metaloproteinasa 11 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Ratas Topo/genética , Ratas Topo/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , ARN/metabolismo , Envejecimiento de la Piel/genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
Nonhealing wounds are a major area of unmet clinical need remaining problematic to treat. Improved understanding of prohealing mechanisms is invaluable. The enzyme arginase1 (ARG1) is involved in prohealing responses, with its role in macrophages best characterized. ARG1 is also expressed by keratinocytes; however, ARG1 function in these critical wound repair cells is not understood. We characterized ARG1 expression in keratinocytes during normal cutaneous repair and reveal de novo temporal and spatial expression at the epidermal wound edge. Interestingly, epidermal ARG1 expression was decreased in both human and murine delayed healing wounds. We therefore generated a keratinocyte-specific ARG1-null mouse model (K14-cre;Arg1fl/fl) to explore arginase function. Wound repair, linked to changes in keratinocyte proliferation, migration, and differentiation, was significantly delayed in K14-cre;Arg1fl/fl mice. Similarly, using the arginase inhibitor N(omega)-hydroxy-nor-L-arginine, human in vitro and ex vivo models further confirmed this finding, revealing the importance of the downstream polyamine pathway in repair. Indeed, restoring the balance in ARG1 activity through the addition of putrescine proved beneficial in wound closure. In summary, we show that epidermal ARG1 plays, to our knowledge, a previously unreported intrinsic role in cutaneous healing, highlighting epidermal ARG1 and the downstream mediators as potential targets for the therapeutic modulation of wound repair.
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Arginasa , Anomalías Cutáneas , Animales , Arginasa/genética , Arginasa/metabolismo , Epidermis/metabolismo , Queratinocitos/metabolismo , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Piel/metabolismo , Anomalías Cutáneas/metabolismoRESUMEN
Endogenous metals are required for all life, orchestrating the action of diverse cellular processes that are crucial for tissue function. The dynamic wound healing response is underpinned by a plethora of such cellular behaviours, occurring in a time-dependent manner. However, the importance of endogenous metals for cutaneous repair remains largely unexplored. Here we combine ICP-MS with tissue-level RNA-sequencing to reveal profound changes in a number of metals, and corresponding metal-regulated genes, across temporal healing in mice. Wound calcium, magnesium, iron, copper and manganese are elevated at 7 days post-wounding, while magnesium, iron, aluminium, manganese and cobalt increase at 14 days post-wounding. At the level of transcription, wound-induced pathways are independently highly enriched for metal-regulated genes, and vice versa. Moreover, specific metals are linked to distinct wound-induced biological processes and converge on key transcriptional regulators in mice and humans. Finally, we reveal a potential role for one newly identified transcriptional regulator, TNF, in calcium-induced epidermal differentiation. Together, these data highlight potential new and diverse roles for metals in cutaneous wound repair, paving the way for further studies to elucidate the contribution of metals to cellular processes in the repair of skin and other tissues.
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OBJECTIVE: To evaluate the effects of multimodal analgesia on postoperative opioid consumption and perioperative pain management in patients undergoing living liver donation. METHODS: A retrospective study was conducted of 129 patients who underwent living liver donation between 2006 and 2015. Patients were separated into 2 cohorts, pre-multimodal analgesia and multimodal analgesia, to allow intergroup analysis. All patients received an intrathecal opioid injection and underwent donor hepatectomy. Primary outcome data compared opioid consumption in oral morphine equivalents for postoperative days (PODs) 0 to 4 between the cohorts. Secondary outcomes compared yearly averaged cumulative opioid consumption on PODs 0 to 4 in oral morphine equivalents; yearly averaged numeric rating scale pain scores; hospital length of stay; and percentage of patients receiving intravenous ketorolac, ketamine, or transversus abdominis plane blocks. RESULTS: For PODs 0 to 4, a 50% reduction in overall opioids administered postoperatively (359 mg vs 179 mg; P<.01) was observed in the multimodal analgesia cohort, whereas no significant difference was found in year-to-year average postoperative pain scores (4.5 vs 3.6). The proportion of patients receiving ketorolac increased to more than 90% by 2013. More than 40% of all patients in the multimodal analgesia group received a perioperative regimen of acetaminophen, gabapentin, ketamine, and transverse abdominal plane blocks (0% in pre-multimodal analgesia). Mean hospital length of stay was reduced from 7.7 to 6.6 days (P<.01). CONCLUSION: Implementation of multimodal analgesia to manage perioperative pain in living liver donation resulted in a 50% reduction of postoperative opioid consumption. Clinically satisfactory average pain scores were maintained for PODs 0 to 4.
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The skin is the body's primary defence against the external environment, preventing infection and desiccation. Therefore, alterations to skin homeostasis, for example with skin ageing, increase susceptibility to skin disease and injury. Skin biological ageing is uniquely influenced by a combination of intrinsic and extrinsic (primarily photoageing) factors, with differential effects on skin structure and function. Interestingly, skin architecture rapidly changes following the menopause, as a direct result of reduced circulating 17ß-estradiol. The traditional clinical benefit of estrogens are supported by recent experimental data, where 17ß-estradiol supplementation prevents age-related decline in the skin's structural and mechanical properties. However, the off-target effects of 17ß-estradiol continue to challenge therapeutic application. Here we discuss how ageing alters the physiological and structural properties of the dermal extracellular matrix, and explore how estrogen receptor-targeted therapies may restore the mechanical defects associated with skin ageing.
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Dermis/metabolismo , Estradiol/metabolismo , Matriz Extracelular/metabolismo , Menopausia/metabolismo , Envejecimiento de la Piel , Femenino , HumanosRESUMEN
Chronic non-healing wounds, which primarily affect the elderly and diabetic, are a significant area of clinical unmet need. Unfortunately, current chronic wound treatments are inadequate, while available pre-clinical models poorly predict the clinical efficacy of new therapies. Here we describe a high throughput, pre-clinical model to assess multiple aspects of the human skin repair response. Partial thickness wounds were created in human ex vivo skin and cultured across a healing time course. Skin wound biopsies were collected in fixative for the whole-mount staining procedure. Fixed samples were blocked and incubated in primary antibody, with detection achieved via fluorescently conjugated secondary antibody. Wounds were counterstained and imaged via confocal microscopy before calculating percentage wound closure (re-epithelialization) in each biopsy. Applying this protocol, we reveal that 2 mm excisional wounds created in healthy donor skin are fully re-epithelialized by day 4-5 post-wounding. On the contrary, closure rates of diabetic skin wounds are significantly reduced, accompanied by perturbed barrier reformation. Combining human skin wounding with a novel whole-mount staining approach allows a rapid and reproducible method to quantify ex vivo wound repair. Collectively, this protocol provides a valuable human platform to evaluate the effectiveness of potential wound therapies, transforming pre-clinical testing and validation.
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Modelos Biológicos , Piel/patología , Coloración y Etiquetado , Cicatrización de Heridas , Anciano , Animales , Anticuerpos/metabolismo , Medios de Cultivo , Diabetes Mellitus/patología , Humanos , Procesamiento de Imagen Asistido por Computador , Indicadores y Reactivos , Reproducibilidad de los Resultados , Piel/lesiones , Cicatrización de Heridas/fisiologíaRESUMEN
Objective: Traditional negative pressure wound therapy (tNPWT) systems can be large and cumbersome, limiting patient mobility and adversely affecting quality of life. PICO™, a no canister single-use system, offers a lightweight, portable alternative to tNPWT, with improved clinical performance. The aim of this study was to determine the potential mechanism(s) of action of single-use NPWT (sNPWT) versus tNPWT. Approach: sNPWT and tNPWT were applied to an in vivo porcine excisional wound model, following product use guidelines. Macroscopic, histological, and biochemical analyses were performed at defined healing time points to assess multiple aspects of the healing response. Results: Wounds treated with single-use negative pressure displayed greater wound closure and increased reepithelialization versus those treated with traditional negative pressure. The resulting granulation tissue was more advanced with fewer neutrophils, reduced inflammatory markers, more mature collagen, and no wound filler-associated foreign body reactions. Of note, single-use negative pressure therapy failed to induce wound edge epithelial hyperproliferation, while traditional negative pressure therapy compromised periwound skin, which remained inflamed with high transepidermal water loss; features not observed following single-use treatment. Innovation: Single-use negative pressure was identified to improve multiple aspects of healing versus traditional negative pressure treatment. Conclusion: This study provides important new insight into the differing mode of action of single-use versus traditional negative pressure and may go some way to explaining the improved clinical outcomes observed with single-use negative pressure therapy.
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Terapia de Presión Negativa para Heridas , Cicatrización de Heridas/fisiología , Animales , Quemaduras/terapia , Humanos , Calidad de Vida , Porcinos , Resultado del TratamientoRESUMEN
Negative pressure wound therapy is a widely used treatment for chronic, nonhealing wounds. Surprisingly, few studies have systematically evaluated the cellular and molecular effects of negative pressure treatment on human skin. In addition, no study to date has directly compared recently available single-use negative pressure modalities to traditional negative pressure devices in a controlled setting. Here we developed a novel large-scale ex vivo human skin culture system to effectively evaluate the efficacy of two different negative pressure wound therapy modalities. Single-use and traditional negative pressure devices were applied to human ex vivo wounded skin sheets cultured over a period of 48 hours. Cellular tissue response to therapy was evaluated via a combination of histological analysis and transcriptional profiling, in samples collected from the wound edge, skin adjacent to the wound, and an extended skin region. Single-use negative pressure wound therapy caused less damage to wound edge tissue than traditional application, demonstrated by improved skin barrier, reduced dermal-epidermal junction disruption and a dampened damage response. Transcriptional profiling confirmed significantly less activation of multiple pro-inflammatory markers in wound edge skin treated with single-use vs traditional negative pressure therapy. These findings may help to explain the greater efficacy of sNPWT in the clinic, while offering a noninvasive system to develop improved NPWT-based therapies.
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Terapia de Presión Negativa para Heridas , Traumatismos de los Tejidos Blandos , Humanos , Piel , Trasplante de Piel , Cicatrización de HeridasRESUMEN
Arguably, the two most important causes of pathological healing in the skin are diabetes and ageing. While these factors have historically been considered independent modifiers of the healing process, recent studies suggest that they may be mechanistically linked. The primary contributor to diabetic pathology is hyperglycaemia, which accelerates the production of advanced glycation end products, a characteristic of ageing tissue. Indeed, advanced age also leads to mild hyperglycaemia. Here, we discuss emerging literature that reveals a hitherto unappreciated link between cellular senescence, diabetes and wound repair. Senescent cells cause widespread destruction of normal tissue architecture in ageing and have been shown to be increased in chronic wounds. However, the role of senescence remains controversial, with several studies reporting beneficial effects for transiently induced senescence in wound healing. We recently highlighted a direct role for senescence in diabetic healing pathology, mediated by the senescence receptor, CXCR2. These findings suggest that targeting local tissue senescence may provide a therapeutic strategy applicable to a broad range of chronic wound types.
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Senescencia Celular , Diabetes Mellitus/fisiopatología , Envejecimiento de la Piel/fisiología , Cicatrización de Heridas/fisiología , Animales , Humanos , Ratones , Receptores de Interleucina-8B/metabolismo , Fenómenos Fisiológicos de la PielRESUMEN
Wound healing is a complex, dynamic process supported by a myriad of cellular events that must be tightly coordinated to efficiently repair damaged tissue. Derangement in wound-linked cellular behaviours, as occurs with diabetes and ageing, can lead to healing impairment and the formation of chronic, non-healing wounds. These wounds are a significant socioeconomic burden due to their high prevalence and recurrence. Thus, there is an urgent requirement for the improved biological and clinical understanding of the mechanisms that underpin wound repair. Here, we review the cellular basis of tissue repair and discuss how current and emerging understanding of wound pathology could inform future development of efficacious wound therapies.
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Cicatrización de Heridas/fisiología , Enfermedad Aguda , Animales , Enfermedad Crónica , Susceptibilidad a Enfermedades , Humanos , Investigación Biomédica Traslacional , Heridas y Lesiones/etiología , Heridas y Lesiones/metabolismo , Heridas y Lesiones/patologíaRESUMEN
Cellular senescence is a fundamental stress response that restrains tumour formation. Yet, senescence cells are also present in non-cancerous states, accumulating exponentially with chronological age and contributing to age- and diabetes-related cellular dysfunction. The identification of hypersecretory and phagocytic behaviours in cells that were once believed to be non-functional has led to a recent explosion of senescence research. Here we discuss the profound, and often opposing, roles identified for short-lived vs. chronic tissue senescence. Transiently induced senescence is required for development, regeneration and acute wound repair, while chronic senescence is widely implicated in tissue pathology. We recently demonstrated that sustained senescence contributes to impaired diabetic healing via the CXCR2 receptor, which when blocked promotes repair. Further studies have highlighted the beneficial effects of targeting a range of senescence-linked processes to fight disease. Collectively, these findings hold promise for developing clinically viable strategies to tackle senescence in chronic wounds and other cutaneous pathologies.
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OBJECTIVE: To determine patient and perioperative characteristics associated with unexpected postoperative clinical deterioration as determined for the need of a postoperative emergency response team (ERT) activation. DESIGN: Retrospective case-control study. SETTING: Tertiary academic hospital. PARTICIPANTS: Patients who underwent general anaesthesia discharged to regular wards between 1 January 2013 and 31 December 2015 and required ERT activation within 48 postoperative hours. Controls were matched based on age, sex and procedure. MAIN OUTCOME MEASURES: Baseline patient and perioperative characteristics were abstracted to develop a multiple logistic regression model to assess for potential associations for increased risk for postoperative ERT. RESULTS: Among 105 345 patients, 797 had ERT calls, with a rate of 7.6 (95% CI, 7.1-8.1) calls per 1000 anaesthetics (0.76%). Multiple logistic regression analysis showed the following risk factors for postoperative ERT: cardiovascular disease (odds ratio [OR], 1.61; 95% CI, 1.18-2.18), neurological disease (OR, 1.57; 95% CI, 1.11-2.22), preoperative gabapentin (OR, 1.60; 95% CI, 1.17-2.20), longer surgical duration (OR, 1.06; 95% CI, 1.02-1.11, per 30 min), emergency procedure (OR, 1.54; 95% CI, 1.09-2.18), and intraoperative use of colloids (OR, 1.50; 95% CI, 1.17-1.92). Compared with control participants, ERT patients had a longer hospital stay, a higher rate of admissions to critical care (55.5%), increased postoperative complications, and a higher 30-day mortality rate (OR, 3.36; 95% CI, 1.73-6.54). CONCLUSION: We identified several patient and procedural characteristics associated with increased likelihood of postoperative ERT activation. ERT intervention is a marker for increased rates of postoperative complications and death.
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Servicio de Urgencia en Hospital/estadística & datos numéricos , Complicaciones Posoperatorias , Estudios de Casos y Controles , Humanos , Tiempo de Internación , Estudios Retrospectivos , Factores de RiesgoAsunto(s)
Aneurisma de la Aorta Abdominal/cirugía , Electrocoagulación/efectos adversos , Síndrome de Fracaso de la Cirugía Espinal Lumbar/terapia , Neuroestimuladores Implantables , Falla de Prótesis/etiología , Estimulación de la Médula Espinal , Anciano , Aneurisma de la Aorta Abdominal/complicaciones , Síndrome de Fracaso de la Cirugía Espinal Lumbar/complicaciones , Humanos , Masculino , Atención Perioperativa , Procedimientos Quirúrgicos VascularesRESUMEN
Promoting cell proliferation is the cornerstone of most tissue regeneration therapies. As platelet-based applications promote cell division and can be customised for tissue-specific efficacy, this makes them strong candidates for developing novel regenerative therapies. Therefore, the aim of this study was to determine if platelet releasate could be optimised to promote cellular proliferation and differentiation of specific tissues. Growth factors in platelet releasate were profiled for physiological and supraphysiological platelet concentrations. We analysed the effect of physiological and supraphysiological releasate on C2C12 skeletal myoblasts, H9C2 rat cardiomyocytes, human dermal fibroblasts (HDF), HaCaT keratinocytes, and chondrocytes. Cellular proliferation and differentiation were assessed through proliferation assays, mRNA, and protein expression. We show that supraphysiological releasate is not simply a concentrated version of physiological releasate. Physiological releasate promoted C2C12, HDF, and chondrocyte proliferation with no effect on H9C2 or HaCaT cells. Supraphysiological releasate induced stronger proliferation in C2C12 and HDF cells compared with physiological releasate. Importantly, supraphysiological releasate induced proliferation of H9C2 cells. The proliferative effects of skeletal and cardiac muscle cells were in part driven by vascular endothelial growth factor alpha. Furthermore, supraphysiological releasate induced differentiation of H9C2 and C2C12, HDF, and keratinocytes. This study provides insights into the ability of releasate to promote muscle, heart, skin, and cartilage cell proliferation and differentiation and highlights the importance of optimising releasate composition for tissue-specific regeneration.
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Plaquetas/citología , Fibroblastos/citología , Regeneración , Adulto , Animales , Diferenciación Celular , Proliferación Celular , Condrocitos , Fibroblastos/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/farmacología , Queratinocitos/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Plasma Rico en Plaquetas , Ratas , Factor A de Crecimiento Endotelial Vascular/metabolismo , Cicatrización de HeridasRESUMEN
Biofilms are prevalent in chronic wounds and once formed are very hard to remove, which is associated with poor outcomes and high mortality rates. Biofilms are comprised of surface-attached bacteria embedded in an extracellular polymeric substance (EPS) matrix, which confers increased antibiotic resistance and host immune evasion. Therefore, disruption of this matrix is essential to tackle the biofilm-embedded bacteria. Here, we propose a novel nanotechnology to do this, based on protease-functionalized nanogel carriers of antibiotics. Such active antibiotic nanocarriers, surface coated with the protease Alcalase 2.4 L FG, "digest" their way through the biofilm EPS matrix, reach the buried bacteria, and deliver a high dose of antibiotic directly on their cell walls, which overwhelms their defenses. We demonstrated their effectiveness against six wound biofilm-forming bacteria, Staphylococcus aureus, Pseudomonas aeruginosa, Staphylococcus epidermidis, Klebsiella pneumoniae, Escherichia coli, and Enterococcus faecalis. We confirmed a 6-fold decrease in the biofilm mass and a substantial reduction in bacterial cell density using fluorescence, atomic force, and scanning electron microscopy. Additionally, we showed that co-treatments of ciprofloxacin and Alcalase-coated Carbopol nanogels led to a 3-log reduction in viable biofilm-forming cells when compared to ciprofloxacin treatments alone. Encapsulating an equivalent concentration of ciprofloxacin into the Alcalase-coated nanogel particles boosted their antibacterial effect much further, reducing the bacterial cell viability to below detectable amounts after 6 h of treatment. The Alcalase-coated nanogel particles were noncytotoxic to human adult keratinocyte cells (HaCaT), inducing a very low apoptotic response in these cells. Overall, we demonstrated that the Alcalase-coated nanogels loaded with a cationic antibiotic elicit very strong biofilm-clearing effects against wound-associated biofilm-forming pathogenic bacteria. This nanotechnology approach has the potential to become a very powerful treatment of chronically infected wounds with biofilm-forming bacteria.