RESUMEN
The fall armyworm (FAW) Spodoptera frugiperda (Smith; Lepidoptera: Noctuidae) is present in over 70 countries in Africa, Asia, and Oceania. Its rapid dispersal since 2016 when it was first reported in western Africa, and associated devastation to agricultural productivity, highlight the challenges posed by this pest. Currently, its management largely relies on insecticide sprays and transgenic Bacillus thuringiensis toxins, therefore understanding their responses to these agents and characteristics of any resistance genes enables adaptive strategies. In Australia, S. frugiperda was reported at the end of January 2020 in northern Queensland and by March 2020, also in northern Western Australia. As an urgent first response we undertook bioassays on two Australian populations, one each from these initial points of establishment. To assist with preliminary sensitivity assessment, two endemic noctuid pest species, Helicoverpa armigera (Hübner; Lepidoptera, Noctuidae) and Spodoptera litura (Fabricius; Lepidoptera, Noctuidae), were concurrently screened to obtain larval LC50 estimates against various insecticides. We characterized known resistance alleles from the VGSC, ACE-1, RyR, and ABCC2 genes to compare with published allele frequencies and bioassay responses from native and invasive S. frugiperda populations. An approximately 10× LC50 difference for indoxacarb was detected between Australian populations, which was approximately 28× higher than that reported from an Indian population. Characterization of ACE-1 and VGSC alleles provided further evidence of multiple introductions in Asia, and multiple pathways involving genetically distinct individuals in Australia. The preliminary bioassay results and resistance allele patterns from invasive S. frugiperda populations suggest multiple introductions have contributed to the pest's spread and challenge the axiom of its rapid 'west-to-east' spread.
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Insecticidas , Mariposas Nocturnas , Animales , Spodoptera/genética , Proteínas Hemolisinas/farmacología , Alelos , Endotoxinas/genética , Resistencia a los Insecticidas/genética , Proteínas Bacterianas/genética , Australia , Insecticidas/farmacología , Larva , Bioensayo , Zea mays/genéticaRESUMEN
The highest natural mortality rate of larval Lepidoptera in field populations occurs in the first instar, but it is highly variable. The pattern and degree of survival is not easily predicted but depends on their ability to establish on host plants. Lepidopteran larval dispersal behaviour, known as 'drop-off', happens when the host is unsuitable for larvae to settle and begin feeding. Understanding drop-off behaviour of Helicoverpa armigera (Hübner) with and without physiological resistance to Bt toxins on Bt and non-Bt cotton plants is an important component for resistance management strategies for this insect. We examined the drop-off behaviour of H. armigera to determine: (1) whether they move the same way or differently in response to Bt and non-Bt, and (2) could H. armigera larvae detect Bt toxin levels in cotton plants or did they move independently of toxin levels? In this study, we assessed the drop-off behaviour of Bt-resistant and Bt-susceptible H. armigera neonates on artificial diets and cotton plants with and without Bt toxin during the first 12 h after hatching. Bt-resistant and Bt-susceptible H. armigera neonates behaved differently on Bt and non-Bt substrates. The percentages of Bt-resistant larvae that dropped off Bt and non-Bt cotton plants were not significantly different. In contrast, significantly more Bt-susceptible larvae dropped off Bt cotton than non-Bt cotton plants over time. Although Bt-susceptible larvae could not detect Bt toxin, they showed preference on non-Bt toxin substrates and were more likely to drop off substrates with Bt toxin.
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Proteínas Hemolisinas , Mariposas Nocturnas , Animales , Larva/fisiología , Proteínas Hemolisinas/farmacología , Proteínas Hemolisinas/genética , Endotoxinas/farmacología , Gossypium , Proteínas Bacterianas/farmacología , Proteínas Bacterianas/genética , Toxinas de Bacillus thuringiensis , Mariposas Nocturnas/fisiología , Plantas Modificadas Genéticamente , Resistencia a los InsecticidasRESUMEN
It is increasingly clear that pest species vary widely in their propensities to develop insecticide resistance. This review uses a comparative approach to analyze the key pest management practices and ecological and biochemical or genetic characteristics of the target that contribute to this variation. We focus on six heliothine species, three of which, Helicoverpa armigera, Heliothis virescens, and Helicoverpa zea, have developed resistances to many pesticide classes. The three others, Helicoverpa punctigera, Helicoverpa assulta, and Helicoverpa gelotopoeon, also significant pests, have developed resistance to very few pesticide classes. We find that host range and movement between alternate hosts are key ecological traits that influence effective selection intensities for resistance. Operational issues are also critical; area-wide, cross-pesticide management practices that account for these ecological factors are key to reducing selection intensity. Without such management, treatment using broad-spectrum chemicals serves to multiply the effects of host plant preference, preadaptive detoxification ability, and high genetic diversity to create a pesticide treadmill for the three high-propensity species.Without rigorous ongoing management, such a treadmill could still develop for newer, more selective chemistries and insecticidal transgenic crops.
Asunto(s)
Insecticidas , Mariposas Nocturnas , Animales , Resistencia a los Insecticidas/genética , Larva , Mariposas Nocturnas/genéticaRESUMEN
BACKGROUND: Helicoverpa armigera and Helicoverpa zea are major caterpillar pests of Old and New World agriculture, respectively. Both, particularly H. armigera, are extremely polyphagous, and H. armigera has developed resistance to many insecticides. Here we use comparative genomics, transcriptomics and resequencing to elucidate the genetic basis for their properties as pests. RESULTS: We find that, prior to their divergence about 1.5 Mya, the H. armigera/H. zea lineage had accumulated up to more than 100 more members of specific detoxification and digestion gene families and more than 100 extra gustatory receptor genes, compared to other lepidopterans with narrower host ranges. The two genomes remain very similar in gene content and order, but H. armigera is more polymorphic overall, and H. zea has lost several detoxification genes, as well as about 50 gustatory receptor genes. It also lacks certain genes and alleles conferring insecticide resistance found in H. armigera. Non-synonymous sites in the expanded gene families above are rapidly diverging, both between paralogues and between orthologues in the two species. Whole genome transcriptomic analyses of H. armigera larvae show widely divergent responses to different host plants, including responses among many of the duplicated detoxification and digestion genes. CONCLUSIONS: The extreme polyphagy of the two heliothines is associated with extensive amplification and neofunctionalisation of genes involved in host finding and use, coupled with versatile transcriptional responses on different hosts. H. armigera's invasion of the Americas in recent years means that hybridisation could generate populations that are both locally adapted and insecticide resistant.
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Genoma de los Insectos , Herbivoria , Mariposas Nocturnas/genética , Animales , Perfilación de la Expresión Génica , Genómica , Especies Introducidas , Larva/genética , Larva/crecimiento & desarrollo , Mariposas Nocturnas/clasificación , Mariposas Nocturnas/crecimiento & desarrollo , Análisis de Secuencia de ADNRESUMEN
Hydroxychloroquine (HCQ; Plaquenil) is used increasingly in the management of a variety of autoimmune disorders, with well established roles in dermatology and rheumatology and emerging roles in oncology. Hydroxychloroquine has demonstrated a survival benefit in patients with systemic lupus erythematosus; some clinicians advocate its use in all such patients. However, Hydroxychloroquine and chloroquine (CQ) have been associated with irreversible visual loss due to retinal toxicity. Hydroxychloroquine retinal toxicity is far more common than previously considered; an overall prevalence of 7.5% was identified in patients taking HCQ for greater than 5 years, rising to almost 20% after 20 years of treatment. This review aims to provide an update on HCQ/CQ retinopathy. We summarise emerging treatment indications and evidence of efficacy in systemic disease, risk factors for retinopathy, prevalence among HCQ users, diagnostic tests, and management of HCQ retinopathy. We highlight emerging risk factors such as tamoxifen use, and new guidance on safe dosing, reversing the previous recommendation to use ideal body weight, rather than actual body weight. We summarise uncertainties and the recommendations made by existing HCQ screening programmes. Asian patients with HCQ retinopathy may demonstrate an extramacular or pericentral pattern of disease; visual field testing and retinal imaging should include a wider field for screening in this group. HCQ is generally safe and effective for the treatment of systemic disease but because of the risk of HCQ retinal toxicity, modern screening methods and ideal dosing should be implemented. Guidelines regarding optimal dosing and screening regarding HCQ need to be more widely disseminated.
Asunto(s)
Hidroxicloroquina/efectos adversos , Retina/efectos de los fármacos , Enfermedades de la Retina/inducido químicamente , Antirreumáticos/efectos adversos , Enfermedades del Tejido Conjuntivo/tratamiento farmacológico , Técnicas de Diagnóstico Oftalmológico , Salud Global , Humanos , Incidencia , Retina/patología , Enfermedades de la Retina/diagnóstico , Enfermedades de la Retina/epidemiología , Factores de RiesgoRESUMEN
We conducted a systematic review of the accuracy of fundus autofluorescence (FAF) imaging for diagnosing and monitoring retinal conditions. Searches in November 2014 identified English language references. Sources included MEDLINE, EMBASE, the Cochrane Library, Web of Science, and MEDION databases; reference lists of retrieved studies; and internet pages of relevant organisations, meetings, and trial registries. For inclusion, studies had to report FAF imaging accuracy quantitatively. Studies were critically appraised using QUADAS risk of bias criteria. Two reviewers conducted all review steps. From 2240 unique references identified, eight primary research studies met the inclusion criteria. These investigated diagnostic accuracy of FAF imaging for choroidal neovascularisation (one study), reticular pseudodrusen (three studies), cystoid macular oedema (two studies), and diabetic macular oedema (two studies). Diagnostic sensitivity of FAF imaging ranged from 32 to 100% and specificity from 34 to 100%. However, owing to methodological limitations, including high and/or unclear risks of bias, none of these studies provides conclusive evidence of the diagnostic accuracy of FAF imaging. Study heterogeneity precluded meta-analysis. In most studies, the patient spectrum was not reflective of those who would present in clinical practice and no studies adequately reported whether FAF images were interpreted consistently. No studies of monitoring accuracy were identified. An update in October 2016, based on MEDLINE and internet searches, identified four new studies but did not alter our conclusions. Robust quantitative evidence on the accuracy of FAF imaging and how FAF images are interpreted is lacking. We provide recommendations to address this.
Asunto(s)
Monitoreo Fisiológico/métodos , Imagen Óptica/métodos , Retina/diagnóstico por imagen , Enfermedades de la Retina/diagnóstico , Fondo de Ojo , Humanos , Reproducibilidad de los ResultadosRESUMEN
PurposeThe aim of this case series is to raise awareness of the emerging issue of serious retinal damage caused by the prolonged use of hydroxychloroquine (HCQ) and the importance of adequate and appropriate monitoring of visual function during treatment.Patient and methodsThis is a small retrospective case series of 3 patients on long-term HCQ who developed serious symptomatic retinal toxicity confirmed on imaging and functional testing.ResultsAll 3 patients were treated with HCQ for over 15 years; two for rheumatoid arthritis (RA), and the third for systemic lupus erythematosus (SLE). All 3 patients had macular involvement varying in severity confirmed with characteristic features on imaging and functional testing (Optical Coherence Tomography (OCT), Autofluorescence (AF) and Humphrey 10-2 visual fields).ConclusionHCQ is widely used to treat autoimmune conditions with a proven survival benefit in patients with SLE. However, long-term use can be associated with irreversible retinal toxicity. These cases highlight that HCQ, like chloroquine, can also cause visual loss in susceptible individuals. Early detection of presymptomatic retinal changes by the introduction of appropriate screening and monitoring is mandatory to limit the extent of irreversible visual loss due to HCQ retinal toxicity.
Asunto(s)
Artritis Reumatoide/tratamiento farmacológico , Diagnóstico Precoz , Hidroxicloroquina/efectos adversos , Lupus Eritematoso Sistémico/tratamiento farmacológico , Retina/patología , Enfermedades de la Retina/inducido químicamente , Campos Visuales , Adulto , Anciano , Antirreumáticos/efectos adversos , Electrorretinografía , Femenino , Angiografía con Fluoresceína , Estudios de Seguimiento , Predicción , Fondo de Ojo , Humanos , Persona de Mediana Edad , Retina/efectos de los fármacos , Enfermedades de la Retina/diagnóstico , Enfermedades de la Retina/fisiopatología , Tomografía de Coherencia Óptica , Pruebas del Campo VisualRESUMEN
In Australia Bt cotton has been planted since 1996, and has greatly improved the control of its key target Helicoverpa armigera (Hübner). There is no strong evidence that genetically modified cotton has been selected for significant physiological resistance to Bt toxin in field populations. There are many possible explanations for the lack of apparent selection that range from high compliance with the resistance management strategy for this technology to a lack of behavioral preference in key traits such as oviposition that could favor survival. To date most experiments that test oviposition of H. armigera on Bt cotton vs. conventional cotton have been done with susceptible moths. We determine the oviposition preference of a field isolated Bt resistant line of H. armigera and a susceptible counterpart when given a choice of non-Bt cotton and Bt-cotton with the same genetic background, and test whether there is any relationship between oviposition site selection (different plant structures) and the survival of the first instar larvae. Within cotton plants, our experiments consistently showed that both resistant and susceptible moths did not choose plants or plant parts that were less toxic in terms of Bt toxin on which to lay eggs. There was one exception in that susceptible moths were more likely to lay eggs on squares of Bt cotton plants than squares of non-Bt cotton. As expected, the mortality of susceptible H. armigera neonates was significantly higher on structures of Bt cotton plants than on those structures of conventional cotton, and survival was greater on flowers than on other structures of Bt cotton. This confirms opportunities for selection for resistance, and demonstrates no advantage in this respect to carrying resistance genes that might overcome the Bt toxins.
Asunto(s)
Mariposas Nocturnas/fisiología , Oviposición , Plantas Modificadas Genéticamente , Animales , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/genética , Conducta Animal , Endotoxinas/genética , Femenino , Gossypium/genética , Proteínas Hemolisinas/genética , Resistencia a los Insecticidas , Larva/fisiología , Control Biológico de VectoresRESUMEN
Cataract surgery was revolutionised by the introduction of modern intraocular lenses in the late 1940's. By the late 1960's to 1970's evidence had emerged that short-wavelength light caused phototoxicity at the retina and retinal pigment epithelium. By the early 1980's ultraviolet filters had been incorporated into intraocular lenses. This caused intense controversy, as there was concern that the UV-filtering chromophore might leach out into the eye causing toxicity. With the arrival of blue-filtering intraocular lenses (BFIOLs) in 1990's, a further debate was ignited as to their safety and potential disadvantages. Selecting the optimal performing intraocular lens to obtain the best visual performance with the fewest potential drawbacks has become complex and challenging for cataract surgeons and their patients with the wide choice of lenses available. Choosing a personalised lens to address astigmatism, presbyopia, spherical aberration, chromatic aberration, and potentially to shield the retina from short-wavelength light is now possible. The potential benefits and possible side effects of these different innovations emphasise the importance of assessing the evidence for their clinical utility, allowing the surgeon and the patient to weigh-up the risk benefit ratio and make an informed decision. The BFIOLs were developed to reduce cyanopsia, address chromatic aberration, and improve contrast sensitivity in different lighting conditions, as well as to prevent short-wavelength light reaching the retina thus potentially reducing the risk of developing age-related macular degeneration. Further design development of the BFIOLs was to mimic the natural crystalline lens absorption and transmittance properties in adulthood. Multiple publications have reported on the potential benefits and pitfalls of implanting a blue-filtering lens. The potential disadvantages raised in the literature over the last 25 years since their introduction, regarding compromise of visual function and disruption of the circadian system, have been largely dispelled. The clear benefits of protecting the retina from short-wavelength light make a BFIOLs a sensible choice. The purpose of this article presented at the Cambridge symposium 2015 is to review the literature on this subject.
Asunto(s)
Materiales Biocompatibles Revestidos , Filtración/instrumentación , Lentes Intraoculares , Protección Radiológica/instrumentación , Rayos Ultravioleta , Extracción de Catarata , Humanos , Implantación de Lentes Intraoculares , Diseño de Prótesis , Traumatismos por Radiación/prevención & control , Trastornos de la Visión/prevención & controlRESUMEN
Peripheral nerve damage is a problem encountered after trauma and during surgery and the development of synthetic polymer conduits may offer a promising alternative to autografts. In order to improve the performance of the polymer to be used for nerve conduits, poly-ε-caprolactone (PCL) films were chemically functionalized with RGD moieties, using a chemical reaction previously developed. In vitro cultures of dissociated dorsal root ganglion (DRG) neurons provide a valid model to study different factors affecting axonal growth. In this work, DRG neurons were cultured on RGD-functionalized PCL films. Adult adipose-derived stem cells differentiated to Schwann cells (dASCs) were initially cultured on the functionalized PCL films, resulting in improved attachment and proliferation. dASCs were also co-cultured with DRG neurons on treated and untreated PCL to assess stimulation by dASCs on neurite outgrowth. Neuron response was generally poor on untreated PCL films, but long neurites were observed in the presence of dASCs or RGD moieties. A combination of the two factors enhanced even further neurite outgrowth, acting synergistically. Finally, in order to better understand the extracellular matrix (ECM)-cell interaction, a ß1 integrin blocking experiment was carried out. Neurite outgrowth was not affected by the specific antibody blocking, showing that ß1 integrin function can be compensated by other molecules present on the cell membrane. Copyright © 2013 John Wiley & Sons, Ltd.
Asunto(s)
Tejido Adiposo/metabolismo , Ganglios Espinales/metabolismo , Neuritas/metabolismo , Oligopéptidos/farmacología , Poliésteres/farmacología , Células Madre/metabolismo , Tejido Adiposo/citología , Animales , Técnicas de Cocultivo , Ganglios Espinales/citología , Masculino , Ratas , Ratas Sprague-Dawley , Células Madre/citologíaRESUMEN
Insecticides impose extreme selective pressures on populations of target pests and so insecticide resistance loci of these species may provide the footprints of 'selective sweeps'. To lay the foundation for future genome-wide scans for selective sweeps and inform genome-wide association study designs, we set out to characterize some of the baseline population genomic parameters of one of the most damaging insect pests in agriculture worldwide, Helicoverpa armigera. To this end, we surveyed nine Z-linked loci in three Australian H. armigera populations. We find that estimates of π are in the higher range among other insects and linkage disequilibrium decays over short distances. One of the surveyed loci, a cytochrome P450, shows an unusual haplotype configuration with a divergent allele at high frequency that led us to investigate the possibility of an adaptive introgression around this locus.
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Variación Genética , Genética de Población , Desequilibrio de Ligamiento , Mariposas Nocturnas/genética , Selección Genética , Animales , Australia , Sistema Enzimático del Citocromo P-450/genética , Femenino , Genes de Insecto , Sitios Genéticos , Haplotipos , Masculino , Modelos Genéticos , Datos de Secuencia Molecular , Análisis de Secuencia de ADNAsunto(s)
Inhibidores de la Angiogénesis/administración & dosificación , Implementación de Plan de Salud/organización & administración , Inyecciones Intravítreas , Programas Nacionales de Salud/organización & administración , Enfermeras Practicantes , Degeneración Macular Húmeda/tratamiento farmacológico , HumanosRESUMEN
Considerable attention has been given to delaying the evolution of insect resistance to toxins produced by transgenic crops. The major pests of cotton in Australia are the Lepidoptera Helicoverpa armigera (Hubner, 1805) and Helicoverpa punctigera (Wallengren), and the toxins deployed in current and imminent transgenic cotton varieties are Cry1Ac, Cry2Ab and Vip3A from Bacillus thuringiensis. In this study, lines that carry alleles conferring resistance to Cry2Ab and Vip3A were isolated using F2 tests. Extensive work on the Cry2Ab resistant lines, and preliminary work on the Vip3A resistant lines, suggested a single common resistance to each toxin in both species thereby justifying the use of more efficient F1 tests as the primary means for monitoring changes over time. A potential further efficiency could be gained by developing a single resistant line that carries both types of Bt resistance. Herein we report on work with both H. armigera and H. punctigera that tests whether dual Cry2Ab-Vip3A resistant lines can be developed and, if so, whether they can be used to effectively monitor resistance frequencies. Furthermore, the creation of dual resistant lines allowed linkage between the Cry2Ab and Vip3A resistances to be investigated for H. punctigera. We show that dual resistant lines can be used to increase the efficiency of the F1 screen for recessive alleles, and that in H. punctigera there is no linkage between Cry2Ab and Vip3A resistance.
Asunto(s)
Proteínas Bacterianas , Endotoxinas , Proteínas Hemolisinas , Resistencia a los Insecticidas/genética , Mariposas Nocturnas/genética , Animales , Toxinas de Bacillus thuringiensis , Femenino , Frecuencia de los Genes , Ligamiento Genético , MasculinoRESUMEN
We have developed a biodegradable polymer scaffold made of a polycaprolactone/polylactic acid (PCL/PLA) film. Surface properties such as topography and chemistry have a vital influence on cell-material interactions. Surface modifications of PCL/PLA films were performed using topographical cues and UV-ozone treatment to improve Schwann cell organisation and behaviour. Schwann cell attachment, alignment and proliferation were evaluated on the grooved UV-ozone treated and non-treated films. Solvent casting of the polymer solution on patterned silicon substrates resulted in films with different groove shapes: V (V), sloped (SL) and square (SQ) shapes. Pitted films, with no grooves, were prepared as a negative control. The UV-ozone treatment was performed to increase hydrophilicity. The process specifications for UV-ozone treatment were evaluated and 5 min radiation time and 6 cm distance to the UV source were suggested as the optimal practise. When cultured on grooved films, Schwann cells elongated on the V and SL shape grooves without crossing over, and grew in the direction of the grooves. However, there was less elongation with more crossing over on the SQ shape grooves. The maximum cell length (511 µm) was observed on the treated V-grooved films. The cells cultured on pitted UV-ozone treated surfaces showed random arrangements with no increase in length. We have demonstrated that the synergic effects of physical cues combined with UV-ozone treatment have the potential to enhance Schwann cell morphology and alignment.
Asunto(s)
Materiales Biocompatibles/química , Polímeros/química , Células de Schwann/citología , Implantes Absorbibles , Animales , Fenómenos Biomecánicos , Adhesión Celular , Proliferación Celular , Forma de la Célula , Células Cultivadas , Materiales Biocompatibles Revestidos/química , Interacciones Hidrofóbicas e Hidrofílicas , Ensayo de Materiales , Microscopía Electrónica de Rastreo , Ozono , Traumatismos de los Nervios Periféricos/terapia , Poliésteres/química , Ratas , Células de Schwann/fisiología , Andamios del Tejido/química , Rayos Ultravioleta , HumectabilidadRESUMEN
Damage to peripheral nerves can cause significant motor or sensory injuries. In serious cases, a nerve is sacrificed from another part of the body to repair a damaged nerve (autograft). The development of biodegradable polymer conduits may offer an alternative to autografts. This study investigated the surface topography and mechanical properties of smooth, pitted and grooved structures of ultra-thin poly (ε-caprolactone)/poly lactic acid blended, solvent-cast films. We have investigated the effect of the groove shape on cell morphology and alignment. Photolithography and dry/wet etching was used to develop patterned silicon substrates with grooves with accurate geometries (V shaped, sloped walls and square shaped). Using a neural cell line (NG108-15), in vitro experiments confirmed good cell attachment and proliferation on all the polymer scaffolds. Imaging techniques demonstrated that there was different cellular responses and morphology according to the shape of the groove. Studies showed that the geometry, particularly the angle of the slope and the space between grooves, affected cellular responses. In addition, biomechanical studies showed that the patterned films had excellent mechanical properties and were stronger than the natural nerve. The conduit tubes were made by rolling the films around a mandrel and using a thermal welding technique to join the edges. The promising biomechanical and in vitro results demonstrate that nerve cell responses are affected by the shape of longitudinal grooves, and particularly by the angle of the slope of the groove walls.
Asunto(s)
Regeneración Tisular Dirigida/instrumentación , Membranas Artificiales , Regeneración Nerviosa/fisiología , Nervios Periféricos/fisiología , Andamios del Tejido , Implantes Absorbibles/efectos adversos , Animales , Adhesión Celular/efectos de los fármacos , Células Cultivadas , Regeneración Tisular Dirigida/efectos adversos , Humanos , Ensayo de Materiales , Regeneración Nerviosa/efectos de los fármacos , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/fisiología , Nervios Periféricos/efectos de los fármacos , Poliésteres/química , Poliésteres/farmacología , Polímeros/química , Polímeros/farmacología , Ratas , Silicio/química , Silicio/farmacología , Propiedades de Superficie , Andamios del Tejido/efectos adversos , Andamios del Tejido/químicaRESUMEN
Two-dimensional (2D) electrospun fibre mats have been investigated as fibrous sheets intended as biomaterials scaffolds for tissue repair. It is recognised that tissues are three-dimensional (3D) structures and that optimisation of the fabrication process should include both 2D and 3D scaffolds. Understanding the relative merits of the architecture of 2D and 3D scaffolds for tendon repair is required. This study investigated three different electrospun scaffolds based on poly(ε-caprolactone) fibres intended for repair of injured tendons, referred to as; 2D random sheet, 2D aligned sheet and 3D bundles. 2D aligned fibres and 3D bundles mimicked the parallel arrangement of collagen fibres in natural tendon and 3D bundles further replicated the tertiary layer of a tendon's hierarchical configuration. 3D bundles demonstrated greatest tensile properties, being significantly stronger and stiffer than 2D aligned and 2D random fibres. All scaffolds supported adhesion and proliferation of tendon fibroblasts. Furthermore, 2D aligned sheets and 3D bundles allowed guidance of the cells into a parallel, longitudinal arrangement, which is similar to tendon cells in the native tissue. With their superior physical properties and ability to better replicate tendon tissue, the 3D electrospun scaffolds warrant greater investigation as synthetic grafts in tendon repair.
Asunto(s)
Regeneración Tisular Dirigida/instrumentación , Poliésteres/química , Traumatismos de los Tendones/cirugía , Andamios del Tejido , Animales , Bovinos , Electroquímica/métodos , Análisis de Falla de Equipo , Regeneración Tisular Dirigida/métodos , Técnicas In Vitro , Ensayo de Materiales , Diseño de Prótesis , Rotación , Traumatismos de los Tendones/patologíaRESUMEN
In vitro characterization of synthetic scaffolds in the laboratory commonly employs sterilization techniques, such as, ultraviolet light or ethanol (EtOH) soaking. These sterilization methods are not sufficient, however, to gain Regulatory approval for therapeutic use. Neglecting the effects medical grade sterilization may have on material properties could lead to years of research never translating to the clinic. The objective of this study was to ascertain whether medical grade gamma irradiation affected the properties of solvent-cast poly(ε-caprolactone)/poly(D, L-lactic acid) blend films for peripheral nerve repair. Scaffolds were sterilized at eight incremental doses of Gamma radiation (0-45 kGy). With increasing radiation dose, tensile testing identified significant reductions in both maximum tensile strength (>40 %) and strain (>90 %); gel permeation chromatography showed a dose-dependent reduction in polymer molecular weight (>46 %) and differential scanning calorimetry highlighted an increase in crystallinity. NG108-15 nerve cells were cultured up to 7 days on gamma irradiated and EtOH soaked films (control). Scanning electron microscopy showed cells proliferated on all films during this time and confirmed cell morphology was unaffected by sterilization method. However, cellular proliferation and number were greater for gamma irradiated films compared to EtOH soaked. Despite material properties being significantly altered, the in vitro response was encouraging and gamma irradiation may prove effective for medical grade sterilization of films intended for peripheral nerve repair.
Asunto(s)
Nervios Periféricos/efectos de los fármacos , Polímeros/farmacología , Animales , Rastreo Diferencial de Calorimetría , Adhesión Celular , Línea Celular Tumoral , Cromatografía en Gel , ADN/metabolismo , Relación Dosis-Respuesta en la Radiación , Rayos gamma , Técnicas In Vitro , Ratones , Microscopía Electrónica de Rastreo , Polímeros/química , Ratas , Espectroscopía Infrarroja por Transformada de Fourier , Propiedades de Superficie , Resistencia a la Tracción , Andamios del TejidoRESUMEN
Cell-material interactions are crucial for cell adhesion and proliferation on biomaterial surfaces. Immobilization of biomolecules leads to the formation of biomimetic substrates, improving cell response. We introduced RGD (Arg-Gly-Asp) sequences on poly-ε-caprolactone (PCL) film surfaces using thiol chemistry to enhance Schwann cell (SC) response. XPS elemental analysis indicated an estimate of 2-3% peptide functionalization on the PCL surface, comparable with carbodiimide chemistry. Contact angle was not remarkably reduced; hence, cell response was only affected by chemical cues on the film surface. Adhesion and proliferation of Schwann cells were enhanced after PCL modification. Particularly, RGD immobilization increased cell attachment up to 40% after 6 h of culture. It was demonstrated that SC morphology changed from round to very elongated shape when surface modification was carried out, with an increase in the length of cellular processes up to 50% after 5 days of culture. Finally RGD immobilization triggered the formation of focal adhesion related to higher cell spreading. In summary, this study provides a method for immobilization of biomolecules on PCL films to be used in peripheral nerve repair, as demonstrated by the enhanced response of Schwann cells.