RESUMEN
INTRODUCTION: Force application (FA) during orthodontic tooth movement is mediated through periodontal ligament (PDL) fibroblasts. FA on deciduous teeth has an inherent risk of root resorption, which is less in permanent teeth. Currently, the root resorption mechanism is poorly understood. We hypothesized that FA alters the morphology and gene expression of PDL fibroblasts. This study was designed to achieve homogenous PDL fibroblast cultures, establish an in-vitro FA model, analyze fibroblast morphology after FA, and compare the gene expressions of PDL fibroblasts of deciduous and permanent teeth after FA. METHODS: Fibroblasts were sorted from primary cultures of deciduous and permanent tooth PDLs. Cell viability was evaluated in the Opticell (Thermo Scientific, Waltham, Mass) FA model. Cellular morphology was analyzed using immunofluorescence staining for actin and focal adhesion complexes. Gene expressions of untreated or pressure-treated PDL fibroblasts of deciduous and permanent teeth were compared by gene array and confirmed by real-time polymerase chain reaction. RESULTS: Cell sorting resulted in cultures containing 98% of PDL fibroblasts. The Opticell model showed 94% cell survival after FA. FA increased fibroblasts' adhesion. Gene arrays and real-time polymerase chain reactions indicated greater up-regulation of DKK2 mRNA in untreated PDL fibroblasts of deciduous teeth and greater up-regulation of ADAMTS1 mRNA in pressurized PDL fibroblasts of deciduous and permanent teeth. CONCLUSIONS: Cell sorting is an efficient method to establish homogenous PDL fibroblast cultures. Using the Opticell FA model allows the maintenance of excellent cell viability. FA increased the surface adherence of fibroblasts. Up-regulation of ADAMTS1 after FA may indicate its involvement in the remodeling of the periodontium during orthodontic tooth movement. Understanding root resorption mechanisms under FA will help to prevent it during orthodontic treatment.
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Fibroblastos/citología , Fibroblastos/fisiología , Expresión Génica , Ligamento Periodontal/citología , Presión , Técnicas de Movimiento Dental , Proteína ADAMTS1/genética , Adolescente , Adulto , Supervivencia Celular , Células Cultivadas , Niño , Citometría de Flujo , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Masculino , ARN Mensajero/genética , Diente/fisiología , Diente Primario/fisiología , Regulación hacia Arriba , Adulto JovenRESUMEN
OBJECTIVE: SS is an autoimmune exocrinopathy affecting â¼1 million patients in the USA that is diagnosed mostly in middle-aged women. Oral fluids (OFs) serving as the mirror of the body were suggested as an ideal non-invasive diagnostic tool. Previously we developed depletion techniques for OF high-abundance proteins to increase visualization of low-abundance proteins. Therefore the aim of this study was to examine the effect of depletion pretreatments on the identification potential of SS OF biomarker candidates. METHODS: Unstimulated OFs were collected from 18 female SS patients and 18 healthy age- and gender-matched controls. High-abundance proteins were depleted using affinity and immunodepletion methodologies followed by semi-quantitative two-dimensional gel electrophoresis and quantitative dimethylation liquid chromatography tandem mass spectrometry (LC-MS/MS). To initially validate the MS results, western blotting was performed. RESULTS: The use of depletion strategy before proteomics analysis increased identification ability by 3-fold. Overall, 79 biomarker candidates were identified. Proteins with the most pronounced fold changes were related to SS serum or tissue factors. In addition, bioinformatics analysis of proteins with a >3-fold increase in SS patients showed calcium-binding proteins, defence-response proteins, proteins involved in apoptotic regulation, stress-response proteins and cell motion-related proteins. Preliminary validation by western blotting of profilin and CA-I indicated similar expression profile trends to those identified by quantitative MS. CONCLUSION: The significance of OF novel depletion methodologies is clearly demonstrated for increased visibility of biomarker candidates as well as for unveiling possible mechanisms involved in this syndrome. This represents a major contribution to our ability to use OF as a future diagnostic fluid.
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Proteínas/metabolismo , Proteómica/métodos , Saliva/metabolismo , Síndrome de Sjögren/diagnóstico , Síndrome de Sjögren/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Biomarcadores/metabolismo , Proteínas de Unión al Calcio/metabolismo , Anhidrasa Carbónica I/metabolismo , Estudios de Casos y Controles , Femenino , Proteínas de Choque Térmico/metabolismo , Humanos , Persona de Mediana Edad , Profilinas/metabolismo , Sensibilidad y EspecificidadRESUMEN
Isolation of highly pure specific cell types is crucial for successful adult stem cell-based therapy. As the number of such cells in adult tissue is low, an extremely efficient method is needed for their isolation. Here, we describe cell-separation methodologies based on magnetic-affinity cell sorting (MACS) MicroBeads with monoclonal antibodies against specific membrane proteins conjugated to superparamagnetic particles. Cells labeled with MACS MicroBeads are retained in a magnetic field within a MACS column placed in a MACS separator, allowing fast and efficient separation. Both positively labeled and non-labeled fractions can be used directly for downstream applications as the separated cell fractions remain viable with no functional impairment. As immunomagnetic separation depends on the interaction between a cell's membrane and the magnetically labeled antibody, separation of specific cells originating from solid tissues is more complex and demands a cell-dissociating pretreatment. In this paper, we detail the use of immunomagnetic separation for the purpose of regenerating damaged salivary gland (SG) function in animal and human models of irradiated head and neck cancer. Each year 500,000 new cases of head and neck cancer occur worldwide. Most of these patients lose SG function following irradiation therapy. SGs contain integrin α6ß1-expressing epithelial stem cells. We hypothesized that these cells can be isolated, multiplied in culture and auto-implanted into the irradiated SGs to regenerate damaged SG function.
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Células Madre Adultas/química , Separación Inmunomagnética/métodos , Integrina alfa6beta1/química , Marcadores de Afinidad/química , Animales , Anticuerpos Monoclonales/química , Membrana Celular/química , Supervivencia Celular , Citometría de Flujo , Neoplasias de Cabeza y Cuello/química , Humanos , Masculino , Ratas , Ratas Sprague-Dawley , Glándulas Salivales/química , Glándulas Salivales/patología , Sensibilidad y EspecificidadRESUMEN
INTRODUCTION: Cardiac troponin (cTn) is the biomarker of choice for detection of myocardial injury. There is a great need for simple point-of-care (POC) troponin testing among patients with chest pain, mainly in the prehospital setting. The purpose of this study was to evaluate the presence of cardiac troponin I (cTnI) in saliva of patients with myocardial injury using alpha-amylase depletion technique. METHODS: Saliva samples were collected from 40 patients with myocardial injury who were tested positive for conventional high-sensitivity cardiac troponin T (cTnT) blood tests, and from 66 healthy volunteers. Saliva samples were treated for the removal of salivary alpha-amylase. Treated and untreated samples were tested with blood cTnI Rapid Diagnostic Test. Salivary cTnI levels were compared to blood cTnT levels. RESULTS: Thirty-six of 40 patients with positive blood cTnT had positive salivary samples for cTnI following alpha-amylase depletion treatment (90.00% sensitivity). Moreover, three of the four negative saliva samples were obtained from patients with relatively low blood cTnT levels of 100 ng/L or less (96.88% sensitivity for 100 ng/L and above). The negative predictive value was 93.65% and rose up to 98.33% considering the 100 ng/L cutoff. Positive predictive values were 83.72% and 81.58%, respectively. Among 66 healthy volunteers and 7 samples yielded positive results (89.39% specificity). CONCLUSION: In this preliminary work, the presence of cTnI in saliva was demonstrated for the first time to be feasibly identified by a POC oriented assay. The specific salivary alpha-amylase depletion technique was shown to be crucial for the suggested assay.
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alfa-Amilasas Salivales , Troponina I , Humanos , Estudios de Factibilidad , Saliva , Troponina T , Biomarcadores , Pruebas en el Punto de AtenciónRESUMEN
OBJECTIVES: This paper is aimed at addressing the urgent need to develop a protocol that will address the operatory and clinical aspects of dental care during the Coronavirus disease 2019 (COVID-19) outbreak. DATA SOURCES: The epidemiology, clinical signs and symptoms, and modes of transmission of COVID-19 are presented. This protocol was established as an international collaboration of three dental universities: Hadassah School of Dental Medicine, Israel; University of Rochester Medical Center, USA; and the University of Pennsylvania, USA. This protocol is based on a detailed review of the existing English language literature as well on the logistic and clinical experience of each facility and the opinion of the authors. The protocol is designed for a hospital setting and includes considerations related to dental treatment in both healthy subjects and those suspected or diagnosed with COVID-19. The first part of this review discusses operatory considerations; the second part discusses general dental clinical aspects; the third part discusses endodontic considerations; and the fourth part discusses surgical aspects. This protocol may be applicable to other future similar pandemics. CONCLUSION: Logistic and clinical steps are required to provide dental care during the COVID-19 outbreak while preventing cross-contamination and protecting the dental team during the provision of care.
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Infecciones por Coronavirus , Coronavirus , Atención Odontológica/tendencias , Endodoncia/tendencias , Transmisión de Enfermedad Infecciosa de Paciente a Profesional , Betacoronavirus , COVID-19 , Infecciones por Coronavirus/epidemiología , Infecciones por Coronavirus/prevención & control , Infecciones por Coronavirus/transmisión , Brotes de Enfermedades , Humanos , Transmisión de Enfermedad Infecciosa de Paciente a Profesional/prevención & control , Israel , Pandemias , Neumonía Viral , SARS-CoV-2RESUMEN
Burning mouth syndrome (BMS) is characterized by a spontaneous and chronic sensation of burning in the oral mucosa, with no apparent signs. The underlying pathophysiological and neuropathic mechanisms remain unclear. Here, we attempt to elucidate some of these mechanisms using proteomic profiling and bioinformatic analyses of whole-saliva (WS) from BMS patients compared to WS from healthy individuals. Qualitative and quantitative proteomic profiling was performed using two dimensional gel electrophoresis (2-DE) and quantitative mass spectrometry (q-MS). In order to improve protein visibility, 21 high abundance proteins were depleted before proteomic profiling. Quantitative proteomic analysis revealed 100 BMS specific proteins and an additional 158 proteins up-regulated by more than threefold in those with BMS. Bioinformatic analyses of the altered protein expression profile of BMS group indicated high correlations to three cellular mechanisms including the neurotrophin signaling pathway. Based on this finding, we suggest that neurotrophin signaling pathway is involved in the pathophysiology of BMS by amplifying P75NTR activity, which in turn increases neural apoptosis thereby reducing sub-papillary nerve fiber density in the oral mucosa.
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Síndrome de Boca Ardiente/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Proteoma/metabolismo , Saliva/metabolismo , Anciano , Anciano de 80 o más Años , Síndrome de Boca Ardiente/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Factores de Crecimiento Nervioso/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteoma/genética , Receptores de Factor de Crecimiento Nervioso/genética , Receptores de Factor de Crecimiento Nervioso/metabolismo , Transducción de SeñalRESUMEN
Salivary glands (SGs) are considered exocrine glands, which mainly secrete water into the oral cavity. Nevertheless, they also exhibit a smaller endocrine secretory pathway toward the bloodstream. The concept of an artificial SG device for exocrine fluid secretion into the oral region in xerostomic patients has been previously studied. The purpose of the current study was to examine the potential of such a device for enhancing bioactive protein secretion. We engineered a plasmid encoding a SG-specific signal peptide sequence adjacent to a normally nonsecreted encoded reporter gene creating a chimera protein, and examined if this construct can enhance secretion from salivary epithelial cells. An N-terminal encoding epidermal growth factor (EGF) sequence was synthesized and inserted into a pGL3 control vector 5' of a firefly luciferase gene, creating a pGL3-EGF signal peptide (pGL3-EGFSP) fused vector. This vector was cotransfected with a pRL-CMV vector containing a Renilla luciferase gene, in 293 cells (serving as controls), and human submandibular gland ductal epithelial (HSG), rat submandibular gland acinar epithelial (SMIE), and rat submandibular gland ductal epithelial (A5) salivary cell lines. The transfected 293, SMIE, and HSG cells showed 8-, 18-, and 40-fold higher luciferase activity, respectively. These observations lead to the concept of an envisioned secretory device, which can serve as a potential biological pump for bioactive proteins.
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Órganos Bioartificiales , Células Epiteliales/citología , Glándula Submandibular/citología , Ingeniería de Tejidos , Animales , Técnicas de Cultivo de Célula , Línea Celular , Factor de Crecimiento Epidérmico/biosíntesis , Factor de Crecimiento Epidérmico/genética , Factor de Crecimiento Epidérmico/metabolismo , Células Epiteliales/metabolismo , Femenino , Expresión Génica , Humanos , Luciferasas de Renilla/biosíntesis , Luciferasas de Renilla/genética , Luciferasas de Renilla/metabolismo , Especificidad de Órganos , Plásmidos/genética , Plásmidos/metabolismo , Señales de Clasificación de Proteína/genética , Ratas , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas y Péptidos Salivales/biosíntesis , Proteínas y Péptidos Salivales/genética , Proteínas y Péptidos Salivales/metabolismo , Glándula Submandibular/metabolismoRESUMEN
PURPOSE: Orthodontically induced inflammatory root resorption (OIIRR) is one of the most prevalent and unavoidable consequence of orthodontic tooth movement. The aim of this study was to discover potential diagnostic protein biomarkers for detection of OIIRR in whole saliva (WS). MATERIAL AND METHODS: Unstimulated WS was collected from 72 subjects: 48 OIIRR patients and 24 untreated, generally healthy, age and gender matched controls. Radiographic assessment of periapical x-rays of four upper incisors taken before and 9 months after bonding was done. High-abundance proteins were depleted followed by two-dimensional-gel-electrophoresis and quantitative mass spectrometry (qMS). Finally, to initially validate qMS results, Western blotting was performed. RESULTS: qMS revealed differentially expressed proteins in the moderate-to-severe OIIRR group, which have never been found in WS before. Additionally, in the moderate-to-severe young OIIRR group, the pathogenetic mechanisms related to actin cytoskeleton regulation and Fc gamma R- mediated phagocytosis were detected, while in adults- to focal adhesion. Preliminary validation by Western blotting of fetuin-A and p21-ARC indicated expression profile trends similar to those identified by qMS. CONCLUSION: The significance of WS novel proteomic methodologies is clearly demonstrated for detecting new OIIRR biomarkers as well as for unveiling possible novel pathogenetic mechanisms in both young and adult patients.
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Proteómica , Resorción Radicular/etiología , Resorción Radicular/metabolismo , Proteínas y Péptidos Salivales/metabolismo , Técnicas de Movimiento Dental/efectos adversos , Biomarcadores/metabolismo , Femenino , Humanos , Inflamación/etiología , Inflamación/metabolismo , MasculinoRESUMEN
Impaired salivary gland (SG) function leading to oral diseases is relatively common with no adequate solution. Previously, tissue engineering of SG had been proposed to overcome this morbidity, however, not yet clinically available. Multiwall inorganic (tungsten disulfide [WS2]) nanotubes (INT-WS2) and fullerene-like nanoparticles (IF-WS2) have many potential medical applications. A yet unexplored venue application is their interaction with SG, and therefore, our aim was to test the biocompatibility of INT/IF-WS2 with the A5 and rat submandibular cells (RSC) SG cells. The cells were cultured and subjected after 1 day to different concentrations of INT-WS2 and were compared to control groups. Growth curves, trypan blue viability test, and carboxyfluorescein succinimidyl ester (CFSE) proliferation assay were obtained. Furthermore, cells morphology and interaction with the nanoparticles were observed by light microscopy, scanning electron microscopy and transmission electron microscopy (TEM), and energy dispersive X-ray spectroscopy. The results showed no significant differences in growth curves, proliferation kinetics, and viability between the groups compared. Moreover, no alterations were observed in the cell morphology. Interestingly, TEM images indicated that the nanoparticles are uptaken by the cells and accumulate in cytoplasmic vesicles. These results suggest promising future medical applications for these nanoparticles.
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Materiales Biocompatibles/farmacología , Fulerenos/farmacología , Ensayo de Materiales/métodos , Nanopartículas/química , Nanotubos/química , Glándula Submandibular/citología , Sulfuros/farmacología , Compuestos de Tungsteno/farmacología , Animales , Proliferación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Nanopartículas/ultraestructura , Nanotubos/ultraestructura , Ratas , Glándula Submandibular/efectos de los fármacos , Glándula Submandibular/ultraestructuraRESUMEN
The ability of elastic fibers to respond to mechanical stimuli suggests that they play a central role in physiological adaptation to external stimuli including application of orthodontic force. The purpose of this study was to examine the effect of external pressure simulating orthodontic force on tropoelastin gene expression in cultured human periodontal ligament fibroblasts (hPDLF). External pressure simulation was achieved by centrifugation for 10, 30, 60, 90 and 120 min of hPDLF in a horizontal microplate rotor. Semi-quantitative RT-PCR analysis of tropoelastin mRNA was performed and beta-actin was used as an internal invariant control. While centrifugal force on mRNA levels of beta-actin showed almost no change, the mRNA levels of tropoelastin increased significantly to a peak level of more than four-fold after 30 min. Thereafter, at 60 min, the mRNA levels remained at more than three-fold. After 90 min, mRNA levels decreased to control levels. The finding that no changes in mRNA levels of beta-actin occurred during the first 90 min of centrifugation validates its use as an invariant control gene in such an experimental model. This study demonstrated that tropoelastin is expressed in hPDLF and that the pressure caused significant time-dependent upregulation of the tropoelastin gene. The responsiveness of the tropoelastin gene to force shows its possible clinical importance in orthodontic tooth movement. Further studies, however, are essential in order to learn whether the high expression of the gene in vitro will also be followed by corresponding protein synthesis and deposition in vivo in the extracellular matrix (ECM) of the periodontal ligament (PDL).
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Ligamento Periodontal/metabolismo , Tropoelastina/metabolismo , Actinas/genética , Actinas/metabolismo , Adolescente , Diente Premolar , Células Cultivadas , Niño , Fibroblastos/metabolismo , Expresión Génica/genética , Humanos , Ortodoncia Correctiva , Presión , ARN Mensajero/metabolismo , Tropoelastina/genéticaRESUMEN
Head and neck cancer is the sixth most common cancer worldwide, resulting in ~ 640,000 cases. Most of these patients have irreversible damage to their salivary glands due to irradiation therapy, which typically leads to significant decrease in quality of life. In the last 2 decades, several strategies have been suggested to overcome this problem; however, no biologically based treatments are available. In the past few years, the authors of the present article and other researchers have focused on a new strategy of re-implantation of autologous salivary gland cells into the residual irradiated salivary glands. This article reviews the current prospective of the irradiation-induced salivary gland impairment mechanisms and the envisioned therapeutic modalities based on stem cell therapy.
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Regeneración/fisiología , Glándulas Salivales/fisiología , Células Madre/fisiología , Autoinjertos/trasplante , Neoplasias de Cabeza y Cuello/radioterapia , Humanos , Traumatismos por Radiación/etiología , Enfermedades de las Glándulas Salivales/etiología , Glándulas Salivales/efectos de la radiación , Trasplante de Células Madre/métodosRESUMEN
Oral fluids (OF) have been suggested as a source of biomarkers for oral and systemic health, but as with other bio-fluids, the presence of high-abundance proteins interferes with the detection of lower-abundance biomarkers. Here, we compared the performance of four depletion treatments: triple depletion (TD) of amylases, albumins and immunoglobulin G; multiple depletion (MD) of amylases and a panel of 20 proteins, a combination of the two (EMD) and combinatorial peptide ligand library based depletion termed CPLL. TD, MD, EMD and CPLL removed 76%, 83%, 85% and 94% of total proteins, respectively, coupled with increased low abundance protein detection and narrowed dynamic range. 2-DE revealed that all depletion pretreatments successfully clarified areas hampered by high-abundance proteins; however, EMD and CPLL exposed the highest number of proteins. Quantitative MS of EMD samples relative to none treated samples indicated that most of downregulated proteins (>90%) were EMD target proteins. In conclusion, a multiple step EMD and CPLL depletion approaches bring about the highest number of protein detection ability and the best hampered-area clearance. As CPLL requires at least 10 fold more protein starting material, we suggest EMD pretreatment as a new detection tool in instances of low protein starting material.
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Biomarcadores/análisis , Proteínas/aislamiento & purificación , Proteómica/métodos , Saliva/química , Adulto , Líquidos Corporales , Técnicas Químicas Combinatorias , Electroforesis en Gel Bidimensional , Humanos , MasculinoRESUMEN
Irradiated head and neck cancer patients suffer from irreversible loss of salivary gland (SG) function, along with significant morbidity and compromised quality of life. To date there is no biologically-based treatment for this distress. Adult salivary gland stem cells are promising candidates for autologous transplantation therapy in the context of tissue-engineered artificial SGs or direct cell therapy. The major restrictions in handling such cells are their limited lifespan during in vitro cultivation, resulting in a narrow time-window for implantation and a risk of tumorigenic changes during culture. To overcome these difficulties, we tested in a rat model the possibility of establishing a personal/autologous SG stem cell bank. SG's integrin-α6ß1-expressing cells were shown to hold a subpopulation of SG-specific progenitor-cells. Explanted and cultured single cell-originated clones were cryopreserved for up to 3 years and shown to exhibit genetic and functional stability similar to noncryopreserved cells, as was emphasized by soft agar assay, division potential assessment, flow cytometric analysis, real-time reverse transcriptase-polymerase chain reaction, in vitro three-dimensional differentiation assay, and immunofluorescence confocal microscopy. Future integration of the novel strategies presented herein to a clinical therapeutic model will allow safe preservation until transplantation and repeated transplantation if needed. These tools open a new venue for adult autologous stem-cell transplantation-based SG regeneration.
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Trasplante de Células/métodos , Neoplasias de Cabeza y Cuello/radioterapia , Glándulas Salivales/citología , Células Madre/citología , Células 3T3 , Animales , Técnicas de Cultivo de Célula , Diferenciación Celular , Criopreservación , Neoplasias de Cabeza y Cuello/terapia , Humanos , Integrina alfa6beta1/biosíntesis , Masculino , Ratones , Microscopía Confocal/métodos , Reacción en Cadena de la Polimerasa/métodos , Ratas , Regeneración , Ingeniería de Tejidos/métodosRESUMEN
Salivary glands (SGs) are irreversibly damaged by irradiation (IR) treatment in head and neck cancer patients. Here, we used an animal irradiation model to investigate and define the molecular mechanisms affecting SGs following IR, focusing on saliva proteome and global transcription profile of submandibular salivary gland (SSG) tissue.We show that saliva secretion was gradually reduced to 50% of its initial level 12 weeks post-IR. Saliva protein composition was further examined by proteomic analysis following mass spectrometry (MS) analysis that revealed proteins with reduced expression originating from SSGs and proteins with increased expression derived from the serum, both indicating salivary tissue damage. To examine alterations in mRNA expression levels microarray analysis was performed. We found significant alterations in 95 genes, including cell-cycle arrest genes, SG functional genes and a DNA repair gene.Tissue damage was seen by confocal immunofluorescence of α-amylase and c-Kit that showed an increase and decrease, respectively, in protein expression. This was coherent with real-time PCR results.This data indicates that IR damages the SSG cells' ability to produce and secrete saliva and proteins, and maintain the physiological barrier between serum and saliva. The damage does not heal due to cell-cycle arrest, which prevents tissue regeneration. Taken together, our results reveal a new insight into IR pathobiology.
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Proteoma/efectos de la radiación , Glándula Submandibular/metabolismo , Glándula Submandibular/efectos de la radiación , Transcriptoma/efectos de la radiación , Animales , Análisis por Conglomerados , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de la radiación , Ratas , Ratas Sprague-Dawley , Saliva/metabolismo , Factores de TiempoRESUMEN
Adult salivary gland stem cells are promising candidates for cell therapy and tissue regeneration in cases of irreversible damage to salivary glands in head and neck cancer patients undergoing irradiation therapy. At present, the major restriction in handling such cells is their relatively limited life span during in vitro cultivation, resulting in an inadequate experimental platform to explore the salivary gland-originated stem cells as candidates for future clinical application in therapy. We established a spontaneous immortal integrin α6ß1-expressing cell line of adult salivary progenitor cells from rats (rat salivary clone [RSC]) and investigated their ability to sustain cellular properties. This line was able to propagate for more than 400 doublings without loss of differentiation potential. RSC could differentiate in vitro to both acinar- and ductal-like structures and could be further manipulated upon culturing on a 3D scaffolds with different media supplements. Moreover, RSC expressed salivary-specific mRNAs and proteins as well as epithelial stem cell markers, and upon differentiation process their expression was changed. These results suggest RSC as a good model for further studies exploring cellular senescence, differentiation, and in vitro tissue engineering features as a crucial step toward reengineering irradiation-impaired salivary glands.
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Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Células Madre Multipotentes/citología , Regeneración , Glándulas Salivales/citología , Glándulas Salivales/fisiología , Animales , Diferenciación Celular , Línea Celular , Separación Celular , Senescencia Celular , Células Epiteliales/citología , Citometría de Flujo , Separación Inmunomagnética , Integrina alfa6beta1/metabolismo , Cariotipificación , Masculino , Microscopía Confocal , Microscopía Fluorescente , Microscopía por Video , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Glándulas Salivales/metabolismo , Ingeniería de Tejidos/métodosRESUMEN
Salivary glands (SGs) secrete more than half a liter of saliva daily. Saliva has many functions in maintaining the normal homeostasis of the oral cavity. Several causes underlie salivary impairment, where irradiation therapy to head and neck cancer patients is one of the most debilitating causes leading to considerable decrease in the patients' quality of life. In the last decade, others and we have focused on implementing tissue engineering principles combined with gene transfer and stem cell methodologies to develop an artificial SG device. This manuscript provides an overview of the current status of engineering an artificial SG.
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Glándulas Salivales , Ingeniería de Tejidos/métodos , Ingeniería de Tejidos/tendencias , Animales , Línea Celular , Humanos , Modelos Biológicos , Técnicas de Cultivo de Órganos , Enfermedades de las Glándulas Salivales/etiología , Enfermedades de las Glándulas Salivales/fisiopatología , Glándulas Salivales/crecimiento & desarrollo , Glándulas Salivales/fisiología , Glándulas Salivales/trasplante , Trasplante de Células Madre/métodosRESUMEN
Regeneration of the salivary glands' (SGs) normal function for patients with cancer of the head and neck treated with irradiation would be a major contribution to their quality of life. This could be accomplished by re-implantation of autologous SG cells into the residual irradiated tissue or by implantation of tissue-engineered artificial SGs. Both methods depend on the isolation of cells able to propagate and differentiate into SG epithelial cells. Recently, it has been shown that SG integrin alpha(6)beta(1)-expressing (SGIE) cells have stem cell capabilities, but these cells could be isolated only after duct ligation insult requiring surgical intervention. Because such an invasive procedure is not clinically acceptable for these patients, our aim in the present study was to explore the use of immuno-magnetic separation of untreated and short heat stress-conditioned rats as a less-insulting methodology for enhancement of these cells. Our results show that submandibular SGIE cells could be isolated and cultivated from untreated animals. However, short heat stress (HS) increased the number of isolated SGIE cells 4.7-fold and their proliferation and clonal capability 4.6-fold and 3 fold, respectively. We believe that SGIE graft cells may be suitable candidates for future tissue-engineered SGs that have been damaged by irradiation in patients with head and neck cancer.
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Integrina alfa6beta1/metabolismo , Glándulas Salivales/citología , Glándulas Salivales/metabolismo , Animales , Proliferación Celular , Separación Celular/métodos , Células Cultivadas , Citometría de Flujo , Inmunohistoquímica , Masculino , Microscopía Electrónica de Transmisión , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Glándulas Salivales/ultraestructura , Ingeniería de Tejidos/métodosRESUMEN
Proteomic characterization of human whole saliva for the identification of disease-specific biomarkers is guaranteed to be an easy-to-use and powerful diagnostic tool for defining the onset, progression and prognosis of human systemic diseases and, in particular, oral diseases. The high abundance of proteins, mainly alpha amylase, hampers the detection of low abundant proteins appearing in the disease state and therefore should be removed. In the present study a 2-DE was used to analyze human whole saliva following the removal of alpha amylase by affinity adsorption to potato starch. After alpha amylase removal whole saliva was analyzed by SDS-PAGE showing at least sixfold removal efficiency and by an alpha amylase activity assay showing 97% reduced activity. MS identification of the captured alpha amylase after elution demonstrated specific removal; 2-DE analysis showed the selective removal of alpha amylase and consequently increased gel resolution. MS identification of protein spots in the 60 kDa area revealed 15 proteins, which were masked before alpha amylase removal. In conclusion, treatment of human whole saliva with an alpha amylase removal device increases gel resolution and enables a higher protein sample for analysis.
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Proteoma/análisis , Proteómica/métodos , Saliva/química , alfa-Amilasas/aislamiento & purificación , Adsorción , Biomarcadores/análisis , Electroforesis en Gel de Poliacrilamida , Humanos , Almidón/químicaRESUMEN
Salivary glands are an accessible organ for gene therapy, enabling expression of recombinant proteins for both exocrine and endocrine secretion. Lentivirus-based vectors have many advantages for gene therapy, including their ability to infect nondividing cells and to stably integrate into the host genome, enabling long-term transgene expression without eliciting an inflammatory immune response. In the present study, murine salivary glands were inoculated with feline immunodeficiency virus (FIV)-based lentiviral vectors expressing various reporter genes. Luciferase expression was observed as early as 24 h posttransduction, peaked at 17-21 days, and remained stable for more than 80 days. Staining with X-gal suggested that mucous acinar cells were effectively transduced. FIV vector transduction with the secreted alkaline phosphatase gene increased serum levels in treated animals for up to 45 days, and the FIV vector harboring the interferon-gamma (IFN-gamma) expression cassette induced an increase in IFN-gamma serum levels as well as in the supernatant of salivary gland explant cultures. These results demonstrate that the transduction of salivary glands with nonprimate lentiviral vectors may provide a novel and highly effective vehicle for long-term endocrine transgene expression.
Asunto(s)
Vectores Genéticos , Lentivirus , Glándulas Salivales/metabolismo , Transducción Genética , Transgenes , Animales , Citomegalovirus/genética , Relación Dosis-Respuesta a Droga , Genes Reporteros , Cinética , Ratones , Regiones Promotoras Genéticas , Glándulas Salivales/citología , Factores de TiempoRESUMEN
The salivary gland has been suggested as an accessible organ for gene transfer to express recombinant proteins locally in the saliva, as well as for secretion to the blood circulation. The aim of this study was to evaluate the efficiency of gene transfer to salivary glands using different viral vectors: adenovirus, vaccinia, herpes simplex type 1 (HSV), and two retroviral vectors (murine leukemia virus (MuLV) and lentivirus). We show, by in situ staining and beta-galactosidase reporter activity assay, that the adenoviral and vaccinia vectors were able to deliver the reporter gene efficiently to acinar and duct cells. The HSV vector was less efficient and infected only the acinar cells. The lentiviral vector infected acinar and duct cells, but at a relatively low efficiency. The MuLV vector did not infect the salivary gland unless cell proliferation was induced. Host immune responses to viral infection, inflammation, apoptosis and lymphocyte infiltration, in the transduced glands, were assessed. The DNA viral vectors induced local lymphocyte infiltration and apoptosis. In contrast, the retroviral vectors did not induce an immune response. Our results describe the outcome of salivary gland infection with each of the five different viral vectors and indicate their advantages and limitations for transferring genes to the salivary glands.