RESUMEN
DESIGN: Time trade-off choice experiment. SETTING: Two large head and neck cancer centres. PARTICIPANTS: Patients who have received treatment for head and neck cancer and members of the head and neck cancer multidisciplinary team. MAIN OUTCOME MEASURES: Participants were asked to rank the outcome scenarios, assign utility values using time trade-off and rate the importance of survival on treatment choice. RESULTS: A total of 49 patients with head and neck cancer and 73 staff members were recruited. Chemoradiotherapy (CRT) optimal outcome was the most preferred health state (34 of 49, 69% patients, and 50 of 73, 68% staff), and CRT with complications was least preferred (27 of 49, 55% patients, and 51 of 73, 70% staff). Using time trade-off, mean utility values were calculated for CRT optimal outcome (0.73 for patients, 0.77 for staff), total laryngectomy (TL) optimal outcome (0.67 for patients, 0.69 for staff), TL outcome with complications (0.46 for patients, 0.51 for staff) and CRT with complications (0.36 for patients, 0.49 for staff). The average survival advantage required for a participant to change their preferred choice was 2.6 years. CONCLUSIONS: We have demonstrated that a significant proportion of patients with head and neck cancer and staff members would not choose CRT to manage locally advanced laryngeal cancer. Staff members rated the health states associated with laryngeal cancer treatment higher than patients who have experienced them, and this is particularly evident when considering the poorer outcomes. The head and neck cancer community should develop methods of practice and decision-making which incorporate elicitation and reporting of patient values as a central principle.
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Actitud , Carcinoma de Células Escamosas/terapia , Toma de Decisiones , Indicadores de Salud , Neoplasias Laríngeas/terapia , Laringectomía/métodos , Calidad de Vida , Adulto , Anciano , Anciano de 80 o más Años , Quimioradioterapia , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Resultado del Tratamiento , Adulto JovenRESUMEN
OBJECTIVE: To critically examine the process of multidisciplinary team (MDT) decision-making with a particular focus on patient involvement. DESIGN: Ethnographic study using direct non-participant observation of 35 MDT meetings and 37 MDT clinics, informal interviews and formal, semistructured interviews with 20 patients and 9 MDT staff members. SETTING: Three head and neck cancer centres in the north of England. PARTICIPANTS: Patients with a diagnosis of new or recurrent head and neck cancer and staff members who attend the head and neck cancer MDT. RESULTS: Individual members of the MDT often have a clear view of which treatment they consider to be 'best' in any clinical situation. When disagreement occurs, the MDT has to manage how it presents this difference of opinion to the patient. First, this is because the MDT members recognise that the clinician selected to present the treatment choice to the patient may 'frame' their description of the treatment options to fit their own view of best. Second, many MDT members feel that any disagreement and difference of opinion in the MDT meeting should be concealed from the patient. This leads to much of the work of decision-making occurring in the MDT meeting, thus excluding the patient. MDT members seek to counteract this by introducing increasing amounts of information about the patient into the MDT meeting, thus creating an 'evidential patient'. Often, only highly selected or very limited information of this type can be available or known and it can easily be selectively reported in order to steer the discussion in a particular direction. CONCLUSIONS: The process of MDT decision-making presents significant barriers to effective patient involvement. If patients are to be effectively involved in cancer decision-making, the process of MDT decision-making needs substantial review.
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Conducta Cooperativa , Toma de Decisiones , Neoplasias de Cabeza y Cuello/terapia , Grupo de Atención al Paciente/organización & administración , Participación del Paciente/métodos , Adulto , Anciano , Manejo de la Enfermedad , Femenino , Neoplasias de Cabeza y Cuello/psicología , Humanos , Entrevistas como Asunto , Masculino , Persona de Mediana Edad , Investigación CualitativaRESUMEN
BACKGROUND: The Cumberland Infirmary, Carlisle, serves a largely remote, rural population of 330 000. The aim of this study was to report the treatment and survival figures for patients treated for laryngeal cancer at this centre. METHODS: The study included 209 consecutive patients with squamous cell carcinoma of the larynx diagnosed between 1996 and 2010 at the Cumberland Infirmary. RESULTS: Disease-specific survival was 100 per cent for stage one, 76 per cent for stage two, 87 per cent for stage three and 46 per cent for stage four. In total, 76 patients (36 per cent) had a laryngectomy, either as primary treatment or as a salvage procedure. CONCLUSION: Our tumour-specific survival rate was very high, and this success may be due in part to high rates of surgical intervention. Survival data compared favourably with other centres, despite less radical radiotherapy regimes. Laryngeal cancer can be managed effectively in a small, relatively remote, multidisciplinary team setting.
Asunto(s)
Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/cirugía , Neoplasias Laríngeas/diagnóstico , Neoplasias Laríngeas/cirugía , Laringectomía , Grupo de Atención al Paciente , Población Rural , Anciano , Carcinoma de Células Escamosas/mortalidad , Carcinoma de Células Escamosas/patología , Supervivencia sin Enfermedad , Femenino , Humanos , Estimación de Kaplan-Meier , Neoplasias Laríngeas/mortalidad , Neoplasias Laríngeas/patología , Laringectomía/métodos , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Estudios Prospectivos , Población Rural/estadística & datos numéricos , Terapia Recuperativa/métodos , Resultado del Tratamiento , Reino UnidoRESUMEN
Gingival enlargement is a fibrotic condition that can arise from systemic administration of the dihydropyridine calcium channel blocker nifedipine. Periostin, a transforming growth factor-beta (TGF-ß)-inducible matricellular protein, has been associated with fibrosis in numerous tissues, but its expression has never been examined in nifedipine-influenced gingival enlargement (NIGE). The objective of this study was to assess if periostin up-regulation is associated with NIGE and whether nifedipine induces periostin expression in gingival fibroblasts. In NIGE tissue (n = 6), periostin is overexpressed in the gingival connective tissue compared with healthy control tissue (n = 6). The transcription factor p-SMAD2/3, which is associated with canonical TGF-ß signaling, localizes to the nuclei in both HGFs and oral epithelial cells in NIGE tissues, but not in control healthy tissue. In vitro culture of HGFs with 30 and 100 ng/mL of nifedipine significantly increased periostin mRNA and protein levels, which correlated with increased levels of active TGF-ß and increased phosphorylation and nuclear localization of SMAD3. Blocking of canonical TGF-ß signaling through inhibition of the TGF-ß receptor I with SB431542 significantly reduced nifedipine-induced SMAD3 phosphorylation and periostin expression. Our results demonstrate that nifedipine up-regulates periostin in HGFs in a TGF-ß-dependent manner.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Moléculas de Adhesión Celular/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Encía/citología , Nifedipino/farmacología , Factor de Crecimiento Transformador beta/efectos de los fármacos , Benzamidas/farmacología , Moléculas de Adhesión Celular/análisis , Técnicas de Cultivo de Célula , Núcleo Celular/efectos de los fármacos , Núcleo Celular/ultraestructura , Tejido Conectivo/metabolismo , Dioxoles/farmacología , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Sobrecrecimiento Gingival/inducido químicamente , Sobrecrecimiento Gingival/metabolismo , Sobrecrecimiento Gingival/patología , Humanos , Fosforilación , Receptores de Factores de Crecimiento Transformadores beta/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Proteína Smad2/análisis , Proteína smad3/análisis , Proteína smad3/efectos de los fármacos , Factor de Crecimiento Transformador beta/análisis , Regulación hacia Arriba/efectos de los fármacosRESUMEN
We aimed to investigate the factors contributing to poor recruitment to the EaStER trial "Early Stage glottic cancer: Endoscopic excision or Radiotherapy" feasibility study. We performed a prospective qualitative assessment of the EaStER trial at three centres to investigate barriers to recruitment and implement changes. Methods used included semi-structured interviews, focus groups and audio-recordings of recruitment encounters. First, surgeons and recruiters did not all accept the primary outcome as the rationale for the trial. Surgeons did not always adhere to the trial eligibility criteria leading to variations between centres in the numbers of "eligible" patients. Second, as both treatments were considered equally successful, recruiters and patients focused on the pragmatics of the different trial arms, favouring surgery over radiotherapy. The lack of equipoise was reflected in the way recruiters presented trial information. Third, patient views, beliefs and preferences were not fully elicited or addressed by recruiters. Fourth, in some centres, logistical issues made trial participation difficult. This qualitative research identified several major issues that explained recruitment difficulties. While there was insufficient time to address these in the EaStER trial, several factors would need to be addressed to launch further RCTs in head and neck cancer. These include the need for clear ongoing agreement among recruiting clinicians regarding details in the study protocol; an understanding of the logistical issues hindering recruitment at individual centres; and training recruiters to enable them to explain the need for randomisation and the rationale for the RCT to patients.
Asunto(s)
Neoplasias Laríngeas , Selección de Paciente , Ensayos Clínicos Controlados Aleatorios como Asunto/métodos , Proyectos de Investigación , Grupos Focales , Personal de Salud , Humanos , Entrevistas como Asunto/métodos , Neoplasias Laríngeas/radioterapia , Neoplasias Laríngeas/cirugía , Prioridad del Paciente , Investigación CualitativaRESUMEN
Connective tissue growth factor (CCN2/CTGF) is not normally expressed in gingival fibroblasts, but is induced by the potent profibrotic cytokine TGFß and is overexpressed in gingival fibrosis. Since CCN2 is a marker and mediator of fibrosis, targeting CCN2 expression in gingival fibroblasts may provide new insights into the future development of novel therapeutic opportunities to treat oral fibrosis. Herein we used real-time polymerase chain-reaction, Western blot, and indirect immunofluorescence analysis to evaluate whether SB-431542, a specific pharmacological inhibitor of TGFß type I receptor (ALK5), blocks the ability of TGFß to induce CCN2 mRNA and protein expression in human gingival fibroblasts. Our results indicate that CCN2 mRNA and protein are induced by TGFß in gingival fibroblasts in a SB-431542-sensitive fashion. These results suggest that blocking ALK5 may be useful in blocking the profibrotic effects of TGFß in gingival fibroblasts.
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Factor de Crecimiento del Tejido Conjuntivo/antagonistas & inhibidores , Fibroblastos/efectos de los fármacos , Encía/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Receptores de Factores de Crecimiento Transformadores beta/antagonistas & inhibidores , Factor de Crecimiento Transformador beta/farmacología , Benzamidas/farmacología , Western Blotting , Técnicas de Cultivo de Célula , Células Cultivadas , Factor de Crecimiento del Tejido Conjuntivo/genética , Dioxoles/farmacología , Fibroblastos/citología , Fibromatosis Gingival/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Encía/citología , Humanos , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/antagonistas & inhibidores , ARN Ribosómico 18S/antagonistas & inhibidores , Receptor Tipo I de Factor de Crecimiento Transformador beta , Factores de Tiempo , Factor de Crecimiento Transformador beta/antagonistas & inhibidoresRESUMEN
Recently identified as a key component of the murine periodontal ligament (PDL), periostin has been implicated in the regulation of collagen fibrillogenesis and fibroblast differentiation. We investigated whether periostin protein is expressed in the human PDL in situ and the mechanisms regulating periostin expression in PDL fibroblasts in vitro. With immunohistochemistry, periostin protein was identified in the PDL, with expression lower in teeth with reduced occlusal loading. In vitro application of uniaxial cyclic strain to PDL fibroblasts elevated periostin mRNA levels, depending on the age of the patient. Treatment with transforming growth factor-beta1 (TGF-ß1) also significantly increased periostin mRNA levels, an effect attenuated by focal adhesion kinase (FAK) inhibition. FAK-null fibroblasts contained no detectable periostin mRNA, even after stimulation with cyclic strain. In conclusion, periostin protein is strongly expressed in the human PDL. In vitro, periostin mRNA levels are modulated by cyclic strain as well as TGF-ß1 via FAK-dependent pathways.
Asunto(s)
Moléculas de Adhesión Celular/análisis , Fibroblastos/efectos de los fármacos , Quinasa 1 de Adhesión Focal/farmacología , Ligamento Periodontal/efectos de los fármacos , Factor de Crecimiento Transformador beta1/farmacología , Adolescente , Adulto , Factores de Edad , Fuerza de la Mordida , Técnicas de Cultivo de Célula , Células Cultivadas , Células del Tejido Conectivo/citología , Fibroblastos/citología , Quinasa 1 de Adhesión Focal/antagonistas & inhibidores , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Ligamento Periodontal/citología , Reacción en Cadena de la Polimerasa/métodos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , ARN Mensajero/análisis , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/antagonistas & inhibidores , Estrés Mecánico , Factor de Crecimiento Transformador beta1/antagonistas & inhibidores , Adulto JovenRESUMEN
OBJECTIVES: To quantify the night-to-night variation in snoring severity; to compare this with inter-subject variation in snoring intensity: to compare multinight mean snoring scores with self-reported subjective scores. DESIGN: Prospective observational study. SETTING: Subjects were recorded during sleep at their own homes. PARTICIPANTS: Twenty patients with socially disruptive snoring awaiting surgery. MAIN OUTCOME MEASURES: Over four consecutive nights using a solid-state sound recording device, the mean, standard deviation and intra-class correlation coefficient were calculated for (a) the loudest 1% of sound, (b) snore frequency and (c) total snore duration. Results were correlated with Snoring Symptom Inventory scores assessed immediately prior to these recordings. RESULTS: Overall mean and intrasubject standard deviation for the loudest 1% of sound was 65.0 (+/-4.1) dB, for snore frequency was 245 (+/-104) per hour and for total snore duration was 4.3% (+/-2.1). Intraclass correlation coefficients were 0.78, 0.74 and 0.67, respectively, suggesting only moderate reliability of these outcome measures. No significant correlation was found between objective and subjective scores for either endpoint. CONCLUSION: Natural night-to-night variation in snoring severity represents a significant proportion of overall snoring variance, thus one night studies of snoring are not reliable. The random error associated with one-night studies exceeds the expected effect size of snoring interventions and so multi-night studies of at least four nights are recommended to reduce the error. However, even multi-night objective measurements correlate poorly with subjective scores of snoring.
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Ritmo Circadiano/fisiología , Monitoreo Fisiológico/métodos , Ronquido/diagnóstico , Adulto , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Estudios Prospectivos , Reproducibilidad de los Resultados , Índice de Severidad de la Enfermedad , Ronquido/fisiopatología , Factores de TiempoRESUMEN
A desirable attribute of implants penetrating epithelium is the inhibition of downward epithelial migration. Simple grooved topographies can inhibit this migration either directly or indirectly by promoting connective tissue attachment, but few studies have focused on the direct effect of geometrically complex topographies on epithelial behavior. Therefore, we examined the influence of novel topographies comprising square floors surrounded by six-sided pillars on periodontal ligament epithelial cell adhesion, morphology, cytoskeletal organization, and migration. Relative to cells on smooth surface, epithelial cells on the pillar substrata adhered closely, exhibited reduced proliferation, had a reduced velocity, but higher persistence. Vinculin staining demonstrated that cells formed mature adhesions on the pillar tops, but smaller punctate adhesion in the gaps and on the pillar walls. Overall more mature adhesions were found on pillars compared to smooth surfaces, which may account for the reduced speed of migration limited on the pillars. F-actin stress fibers were predominantly found on pillar tops within 6 h, whereas microtubules (MTs) had a tendency to form in the gaps between the six-sided pillars. In conclusion, microfabricated pillars altered epithelial migration in ways that could prove useful in inhibition of epithelial downward migration on transmucosal implants.
Asunto(s)
Adhesión Celular , Citoesqueleto/metabolismo , Células Epiteliales/citología , Ingeniería de Tejidos , Andamios del Tejido/química , Animales , Movimiento Celular , Proliferación Celular , Células Cultivadas , Células Epiteliales/metabolismo , Ligamento Periodontal/citología , Propiedades de Superficie , PorcinosRESUMEN
Anaerobic digestion of animal waste is a technically viable process for the abatement of adverse environmental impacts caused by animal wastes; however, widespread acceptance has been plagued by poor economics. This situation is dismal if the technology is adapted for treating low strength animal slurries because of large digester-volume requirements and a corresponding high energy input. A possible technology to address these constraints is the anaerobic sequencing batch reactor (ASBR). The ASBR technology has demonstrated remarkable potential to improve the economics of treating dilute animal waste effluents. This paper presents preliminary data on the effects of temperature and frequency-cycle on the operation of an ASBR at a fixed hydraulic retention time (HRT). The results suggest that within the parameter range under consideration, temperature did not affect the biogas yield significantly, however, higher cycle-frequency had a negative effect. The biogas quality (%CH(4)) was not significantly affected by temperature nor by the cycle-frequency. The operating principle of the ASBR follows four phases: feed, react, settle, and decant in a cyclic mode. To improve the biogas production in an ASBR, one long react-phase was preferable compared to three shorter react-phases. Treatment of dilute manure slurries in an ASBR at 20 degrees C was more effective than at 35 degrees C; similarly more bio-stable effluents were obtained at low cycle-frequency. The treatment of dilute swine slurries in an ASBR at the lower temperature (20 degrees C) and lower cycle-frequency is, therefore, recommended for the bio-stabilization of dilute swine wastewaters. The results also indicate that significantly higher VFA degradation occurred at 20 degrees C than at 35 degrees C, suggesting that the treatment of dilute swine slurries in ASBRs for odor control might be more favorable at the lower than at the higher temperatures examined in this study. Volatile fatty acid reduction at the two reactor temperatures and cycle-frequencies, from a high of 639+/-75 mg/L to a low of 92+/-23 mg/L, greatly reduced the odor and the odor-generation potential in post-treatment storage. The nutrients (both N and P) in the waste influent were conserved in the effluents.
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Bacterias Anaerobias/metabolismo , Residuos Industriales , Agricultura/métodos , Animales , Biomasa , Reactores Biológicos , Biotecnología/métodos , Diseño de Equipo , Estiércol , Aguas del Alcantarillado , Porcinos , Temperatura , Eliminación de Residuos Líquidos , Purificación del AguaRESUMEN
BACKGROUND: The use of stitches and staples for the closure of surgical wounds is associated with complications for both the surgeon and the patient. Histoacryl (butyl1-2cyanoacrylate glue) is widely used for wound closure in Accident and Emergency departments, in particular for facial and scalp wounds. METHOD: We have used Histoacryl for closure of various surgical incisions in a series of 50 cases and assessed the cosmetic outcome at three to four weeks. RESULTS: There were no wound related complications in the form of infection or dehiscence. In one case however, there was formation of synechiae in the external auditory meatus following an endaural incision. CONCLUSION: We recommend the use of Histoacryl over traditional closure using skin suture material in otological surgery. Our experience was also successful in parotid cases.
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Enfermedades del Oído/cirugía , Enbucrilato/uso terapéutico , Procedimientos Quirúrgicos Otológicos/métodos , Dehiscencia de la Herida Operatoria/prevención & control , Técnicas de Sutura/instrumentación , Estudios de Seguimiento , Humanos , Resultado del TratamientoRESUMEN
AIM: To compare voice performance following Bioplastique injection with that following Isshiki thyroplasty. MEASURES: A 12 item, self-reported voice performance questionnaire was completed and observer-rated perceptual voice analysis scores were also measured, before and after Bioplastique injection, for 14 patients. Results were compared with our previously reported outcomes for 28 thyroplasty patients. RESULTS: Only 14 of 30 patients had complete datasets. For these patients, the mean pre-operative voice performance questionnaire score improved significantly, from 40.3 to 18.9 (p=0.002, Wilcoxon test). All perceptual analysis parameters showed significant improvement. These results compare favourably with the thyroplasty cohort (mean voice performance questionnaire score: pre-operative 35; post-operative 18; p<0.001). One Bioplastique patient developed contralateral paresis, requiring partial removal of the material 18 months later. Two thyroplasty patients experienced complications and three required revision. CONCLUSIONS: Both Bioplastique injection and Isshiki thyroplasty resulted in a significant improvement in both subjective and perceptual voice performance. Our data suggest that the effect size of the two interventions is approximately similar (in nonrandomised cohorts of surviving patients). As in many similar studies, the incomplete follow-up data reflect severe comorbidity. Bioplastique injection is a quicker procedure associated with fewer complications, and thus appears superior to framework surgery in patients with limited life expectancy.
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Polímeros/administración & dosificación , Prótesis e Implantes , Cartílago Tiroides/cirugía , Parálisis de los Pliegues Vocales/terapia , Calidad de la Voz , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Encuestas y Cuestionarios , Parálisis de los Pliegues Vocales/cirugíaRESUMEN
The fabrication of surfaces that stimulate increased adhesion, migration, and differentiated function of osteoblasts has been viewed as being desirable for many orthopedic applications. Previous studies have shown that microfabricated pits and grooves alter adhesion, spreading, matrix secretion, and production of mineral by rat calvarial osteoblasts (RCOs). The mechanisms underlying these effects are unknown, although microenvironment and cell alignment are considered to play a role. The aim of this work was to investigate the behavior of RCOs on microfabricated discontinuous-edge surfaces (DESs), which could provide an alternative means to control both the microenvironment and cellular alignment. Two types of discontinuous-type structures were employed, gap-cornered boxes and micron scale pillars. DES gap-cornered boxes and the pillars influenced the arrangement of F-actin, microtubules, and vinculin. Osteoblasts were guided in their direction of migration on both types of substrata. Both box DESs and pillars altered the staining intensity and localization pattern of phosphotyrosine and src-activated FAK localization. Cell multilayering, matrix deposition, and mineralization were enhanced on both discontinuous topographies when compared with smooth controls. This study shows that DESs alter adhesion, migration, and proliferative responses from osteoblasts at early time points (<1 week) and promote multilayering, matrix deposition, and mineral deposition at later times (2-6 weeks). Such topographical patterns could potentially be employed as effective surface features on bone-contacting implants or in membrane-based periodontal applications.
Asunto(s)
Matriz Ósea/metabolismo , Regeneración Ósea/fisiología , Calcificación Fisiológica/fisiología , Citoesqueleto/metabolismo , Osteoblastos/metabolismo , Osteogénesis/fisiología , Actinas/metabolismo , Actinas/ultraestructura , Fosfatasa Alcalina/metabolismo , Animales , Matriz Ósea/ultraestructura , Adhesión Celular/fisiología , Movimiento Celular/fisiología , Proliferación Celular , Células Cultivadas , Citoesqueleto/ultraestructura , Resinas Epoxi/uso terapéutico , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Microscopía por Video , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Osteoblastos/ultraestructura , Fosforilación , Fosfotirosina/metabolismo , Ratas , Cráneo/metabolismo , Cráneo/ultraestructura , Vinculina/metabolismo , Vinculina/ultraestructuraRESUMEN
Cell adhesion, shape, and directed migration are some of the fundamental processes underlying tissue development and organization. The setting of geometric limits on cellular behavior has led to the hypothesis that a continuous edge is required to elongate a cell and guide its direction of movement. The aim of this study was to examine the validity of this hypothesis by examining the response of human gingival fibroblasts and periodontal ligament epithelial cells, to microfabricated surfaces that incorporate discontinuous edges. Cell response was assessed through spreading, morphology, cytoskeletal organization, and time-lapse microscopy, on substrata with a pattern of repeated open boxes with gaps at the corners. Fibroblasts attached and spread within 6 h, adopting either a square, triangular, or diagonally elongated morphology. Epithelial cells took longer to adhere, but were observed to adopt morphologies similar to those of the fibroblasts. Addition of colcemid or cytochalasin-D attenuated the orientation and alignment of both fibroblasts and epithelial cells. Fibroblasts and epithelial cell migration was guided diagonally in their movement through gaps in the square pattern, demonstrating that a continuous edge is not a prerequisite for guided cell migration.
Asunto(s)
Células Epiteliales/fisiología , Fibroblastos/fisiología , Animales , Técnicas de Cultivo de Célula/instrumentación , Movimiento Celular/fisiología , Células Cultivadas , Citocalasina D/farmacología , Citoesqueleto/efectos de los fármacos , Citoesqueleto/fisiología , Citoesqueleto/ultraestructura , Demecolcina/farmacología , Células Epiteliales/citología , Células Epiteliales/ultraestructura , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/ultraestructura , Humanos , PorcinosRESUMEN
Understanding the response of chondrocytes to topographical cues and chemical patterns could provide invaluable information to advance the repair of chondral lesions. We studied the response of primary chondrocytes to nano- and micro-grooved surfaces, and sulphated hyaluronic acid (HyalS). Cells were grown on grooves ranging from 80 nm to 9 microm in depth, and from 2 microm to 20 microm in width. Observations showed that the cells did not spread appreciably on any groove size, or alter morphology or F-actin organization, although cells showed accelerated movement on 750 nm deep grooves in comparison to flat surfaces. On chemical patterns, the cells migrated onto, and preferentially attached to, HyalS and showed a greater degree of spreading and F-actin re-arrangement. This study shows that 750 nm deep grooves and sulphated hyaluronic acid elicit responses from primary chondrocytes, and this could have implications for the future direction of cartilage reconstruction and orthopaedic treatments in general.
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Cartílago Articular/citología , Condrocitos/citología , Ácido Hialurónico/farmacología , Actinas/metabolismo , Animales , Materiales Biocompatibles , Adhesión Celular/fisiología , Técnicas de Cultivo de Célula , Movimiento Celular/fisiología , Condrocitos/efectos de los fármacos , Condrocitos/ultraestructura , Citoesqueleto/metabolismo , Ácido Hialurónico/análogos & derivados , Microscopía de Fuerza Atómica , Ovinos , Propiedades de Superficie/efectos de los fármacosRESUMEN
The isolation and culture of articular chondrocytes is a prerequisite of their use in tissue engineering, but prolonged culture and passaging is associated with de-differentiation. In this paper we studied the influence of nanometric and micrometric grooves (85 nm to 8 microm in depth and 2 microm to 20 microm in width) on 1st and 2nd passage ovine chondrocytes since our earlier findings indicate that primary cells are not affected by such features. 1st and 2nd passage chondrocytes cultured on grooved substrata showed a polarisation of cell shape parallel to the groove long axis and F-actin condensations were evident at groove ridge boundaries. An increase in cell migration with increasing groove depth was observed. Both passages of chondrocytes maintained type II collagen expression, but to a lesser degree in 2nd. This study demonstrates that passage number alters the response of chondrocytes to micrometric and nanometric topography, and could be important in ex vivo cartilage engineering.
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Cartílago Articular/citología , Movimiento Celular , Condrocitos/citología , Citoesqueleto/metabolismo , Microquímica , Nanotecnología , Actinas/metabolismo , Animales , Adhesión Celular , Proliferación Celular , Forma de la Célula , Condrocitos/fisiología , Colágeno Tipo II/metabolismo , Citoesqueleto/química , Microscopía de Interferencia , Fenotipo , Ovinos , Factores de TiempoRESUMEN
Ca2+ channels at the plasma membrane of stomatal guard cells contribute to increases in cytosolic free [Ca2+] ([Ca2+](i)) that regulate K+ and Cl- channels for stomatal closure in higher-plant leaves. Under voltage clamp, the initial rate of increase in [Ca2+](i) in guard cells is sensitive to the extracellular divalent concentration, suggesting a close interaction between the permeant ion and channel gating. To test this idea, we recorded single-channel currents across the Vicia guard cell plasma membrane using Ba2+ as a charge carrying ion. Unlike other Ca2+ channels characterised to date, these channels activate at hyperpolarising voltages. We found that the open probability (P(o)) increased strongly with external Ba2+ concentration, consistent with a 4-fold cooperative action of Ba2+ in which its binding promoted channel opening in the steady state. Dwell time analyses indicated the presence of a single open state and at least three closed states of the channel, and showed that both hyperpolarising voltage and external Ba2+ concentration prolonged channel residence in the open state. Remarkably, increasing Ba2+ concentration also enhanced the sensitivity of the open channel to membrane voltage. We propose that Ba2+ binds at external sites distinct from the permeation pathway and that divalent binding directly influences the voltage gate.
Asunto(s)
Bario/metabolismo , Canales de Calcio/fisiología , Fabaceae/fisiología , Activación del Canal Iónico , Plantas Medicinales , Membrana Celular/metabolismo , Membrana Celular/fisiología , Fabaceae/metabolismo , Fabaceae/ultraestructura , Técnicas de Placa-Clamp , Hojas de la Planta/metabolismo , Hojas de la Planta/fisiología , Hojas de la Planta/ultraestructura , Protoplastos/metabolismo , Protoplastos/fisiología , Protoplastos/ultraestructuraRESUMEN
The rat Crisp-1 gene encodes Protein DE (acidic epididymal glycoprotein; AEG), a glycoprotein secreted by the epididymal epithelium that associates with maturing sperm and has been implicated in the process of sperm-egg fusion. Previous characterization of the Crisp-1 messenger RNA in the rat epididymis has demonstrated the presence of 3 splice variants (Klemme et at, 1999). This study was undertaken to determine if expression of the Crisp-1 splice variants in the rat epididymis is region-specific and correlates with the region-specific pattern of synthesis of the D and E forms of the Crisp-1 protein. Expression of each of the splice variants was shown by RNase protection assays to be under the control of androgens, but they are not differentially regulated either within the epididymal segments or along the length of the organ. The reported structure of the mouse Crisp-1 gene does not include an exon that is equivalent to the rat exon 1, suggesting that the rat splice variants cannot exist in the mouse and may be specific to the rat. Furthermore, the mouse transcription start site is situated in a different region of the gene than in the rat. In this study, a comparison of the mouse and rat genes in the region flanking the mouse exon 1 and the rat exon 2 (within the rat intron 1) shows greater than 80% sequence identity, including the conservation of several putative androgen receptor binding sites. In addition, the rat gene is shown to have a corrupted TATA box in intron 1 that corresponds to the TATA box located in the mouse gene. These observations explain the preferential transcription for the mouse gene in this region, while the predominant start site for the rat gene is 5' of the upstream exon 1. Although an exon corresponding to the rat exon 1 has not been found in the mouse gene, reverse transcription-polymerase chain reaction experiments using mouse epididymal RNA suggest that such an exon exists in the mouse gene and is transcribed at low frequency.
Asunto(s)
Empalme Alternativo/genética , Epidídimo/fisiología , Metaloproteínas/genética , Hormonas Testiculares/genética , Animales , Secuencia de Bases , Clonación Molecular , Proteínas Secretorias del Epidídimo , Exones , Regulación de la Expresión Génica/genética , Intrones , Masculino , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Secuencias Reguladoras de Ácidos Nucleicos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Especificidad de la Especie , Transcripción Genética/genéticaRESUMEN
In stomatal guard cells of higher-plant leaves, abscisic acid (ABA) evokes increases in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) by means of Ca(2+) entry from outside and release from intracellular stores. The mechanism(s) for Ca(2+) flux across the plasma membrane is poorly understood. Because [Ca(2+)](i) increases are voltage-sensitive, we suspected a Ca(2+) channel at the guard cell plasma membrane that activates on hyperpolarization and is regulated by ABA. We recorded single-channel currents across the Vicia guard cell plasma membrane using Ba(2+) as a charge-carrying ion. Both cell-attached and excised-patch measurements uncovered single-channel events with a maximum conductance of 12.8 +/- 0.4 pS and a high selectivity for Ba(2+) (and Ca(2+)) over K(+) and Cl(-). Unlike other Ca(2+) channels characterized to date, these channels rectified strongly toward negative voltages with an open probability (P(o)) that increased with [Ba(2+)] outside and decreased roughly 10-fold when [Ca(2+)](i) was raised from 200 nM to 2 microM. Adding 20 microM ABA increased P(o), initially by 63- to 260-fold; in both cell-attached and excised patches, it shifted the voltage sensitivity for channel activation, and evoked damped oscillations in P(o) with periods near 50 s. A similar, but delayed response was observed in 0.1 microM ABA. These results identify a Ca(2+)-selective channel that can account for Ca(2+) influx and increases in [Ca(2+)](i) triggered by voltage and ABA, and they imply a close physical coupling at the plasma membrane between ABA perception and Ca(2+) channel control.