Wir danken unseren Kollegen in RIKEN-BSI für die freundliche Unterstützung und fruchtbare Ratschläge bei dieser Arbeit. Ich möchte meinen aufrichtigen Dank Professor Shigemi Mori, Professor G bor Juh sz, Drs. auszudrücken. Kiyoji Matsuyama, Katalin AK kesi und rp d Dobolyi, die mich zu Mikrodialyse-Methode während der Diplom-Studium an der Graduate University for Advanced Studies, Japan-Studie beraten. Abschließend danken wir Herrn Chris Sarnecky und Dr. Akitsugu Takasu für ihre Unterstützung in Video-Aufzeichnung.

Schritt 1. Vorbereitung der Sonde Implantation <ol><li> Anästhesie (nicht im Video gezeigt) Anesthetize jeder Vogel mit einem interperitoneale Injektion eines Ketamin (12,5 mg / kg Körpergewicht) und Xylazin (25 mg / kg) Mischung auf einem konstanten Niveau von chirurgischen Anästhesie zu erzeugen. </li><li> Fix einen Kopf zu einem stereotaktischen Rahmen Verwenden Sie Xylocain (TM) Gelee zu lokalen Schmerzen Ohr Bars &quot;Druck zu reduzieren. Der Kopf des Vogels ist in einem stereotaktischen Apparatur, bestehend aus Ohr Bars und einen Schnabel Halter, dass der Kopf in einem Winkel von 45 ° hält fixiert. Während der stereotaktischen Operation zur Implantation, erwärmen den Körper des Vogels durch chirurgische Pad mit thermischen (Temperatur = 34 ~ 36 ° C). </li><li> Desinfizieren OP-Bereich Desinfizieren Sie das OP-Feld mit isodine (TM) vor der Operation. </li></ol> Schritt 2. Stereotaktische Positionierung <ol><li> Referenzpunkt Ausrichtung Richten Sie Dummy-Sonde (Glasmikropipette), um die Lambda (Y-Punkt) und Mark Y-Punkt mit dem Farbstoff gefüllten Pipette. Verbleibende Dextran-TMR der letzten Tracer-Studie wurde in dem Video verwendet. </li><li> Inzision der Haut Entfernen Sie Haut und setzen der Oberfläche des Schädels. Dann reinigen Sie die Oberfläche des Schädels mit einem Wattestäbchen. </li><li> Die Positionierung der Dummy-Kanüle (in horizontaler Richtung) Bewegen Sie den Dummy-Kanüle an den Zielort (AP-Achse und Lateral-Koordination) </li><li> Kleine Kraniotomie und Inzision der Dura mater. Verwenden einer feinen Pinzette für Kraniotomie und einer Geldstrafe Injektionsnadel (30 G) für Einschnitte der Dura mater. Dann reinigen Sie die Oberfläche des Schädels. </li><li> Endgültige Positionierung (Implantation) von Dummy-Kanüle Suchen Sie die Spitze des Führungskanüle auf 0,30 mm über dem Mikrodialysesonde-anvisierten Region. Schließlich ist die Oberfläche des Schädels wieder, denn eine saubere und trockene Oberfläche für den harten Bindung erforderlich ist. </li></ol> Schritt 3. Eine Vorbehandlung der Oberfläche der Schädelbasis (Die ähnliche Vorgehensweise für &quot;direkte Befüllung Wiederherstellung&quot; der zahnärztlichen Produkt wird eingesetzt werden.) <ol><li> Primer-Behandlung <ol><li> Mix PRIMAR Flüssigkeit A mit B auf einem Teller. </li><li> Übernehmen Sie die Mischung auf der Oberfläche des Schädels mit einem kleinen Pinsel. </li><li> Lassen Sie für 30 Sekunden. </li><li> Trocken mit milder Luftstrom. (Nicht waschen.) </li></ol></li><li> Die Anwendung des Bindemittels <ol><li> Wenden Sie das Bindemittel an der Oberfläche des Schädels mit einem kleinen Pinsel. </li><li> Tragen Sie einen sanften Luftstrom. (Nicht waschen.) </li><li> Light-Kur für 20 Sekunden. </li></ol></li></ol> Schritt 4. Montage Komposit <ol><li> Berg-Harz mit einfachen plugger Wenn ein Plain Stopfer nicht verfügbar ist, kann ein Mikro-Spatel verwendet werden. </li><li> Lichthärtung </li><li> Incremental Montage Incremental Montage zu gewährleisten, eine vollständige Umsetzung des Harzes empfohlen. Bei inkrementellen Montage gelten Haftvermittler vor jeder Montage. </li><li> Mold den Komposit-und lichthärtenden für die endgültige Aushärtung. </li><li> Ende der Operation </li></ol> Reinigen Sie den OP-Bereich, gelten Antibiotikum (Gentamicin Salbe), und entfernen Sie das Tier aus der stereotaktischen Flamme. Dann halten die Tiere in einem thermo-kontrollierten Kammer (Temperatur = 33-35 ° C) für post-Aufwachraum. Wie in dem Video zu sehen, wurde kein anormales Verhalten in einem der Tiere am nächsten Tag nach der Operation beobachtet.

<ol>
	<li>Adell, A., Artigas, F., Boulton, A., Baker, G., Bateson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+Brain+microdialysis:+Principles+and+applications.">In vivo Brain microdialysis: Principles and applications.</a> In Vivo Neuromethods: Neuromethods. 32, 325-357 (1998).</li><li>Kendrick, K., Robinson, T., Justice, J., Bateson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microdialysis+in+large+unrestrained+animals:+neuroendocrine+and+behavioral+studies+of+acetylcholine,+amino+acid,+monoamine+and+neuropeptide+release+in+the+sheep.">Microdialysis in large unrestrained animals: neuroendocrine and behavioral studies of acetylcholine, amino acid, monoamine and neuropeptide release in the sheep.</a> Microdialysis in the Neuroscience. 7, 327-348 (1991).</li><li>Oakley, B., Schafer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Experimental Neurobiology: A laboratory Manual. , (1978).</li><li>Okumura, T., Yamashita, Y., Okanoya, K., Tani, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+of+the+sensori-motor+nucleus+NIf+in+generation+of+complex+syntactical+song+in+the+Bengalese+Finch+I.+A+Biological+study.">Function of the sensori-motor nucleus NIf in generation of complex syntactical song in the Bengalese Finch I. A Biological study.</a> Soc. Neurosci Abst. 33, (2007).</li><li>Boulton, A., Baker, G., Robinson, T., Bateson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relation+of+brain+electrical+activity+to+behavior.">The relation of brain electrical activity to behavior.</a> In Vivo Neuromethods: Neuromethods. 32, 325-357 (1998).</li><li>Yamashita, Y., Okumura, T., Okanoya, K., Tani, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+of+the+sensori-motor+nucleus+NIf+in+generation+of+complex+syntactical+song+in+the+Bengalese+finch.">Function of the sensori-motor nucleus NIf in generation of complex syntactical song in the Bengalese finch.</a> , 101-104 (2007).</li></ol>

Die Experimente wurden in Übereinstimmung mit den Richtlinien für Tierversuche der Brain Science Institute, RIKEN (RIKEN-BSI) und des Instituts Tier Ethikkommission genehmigt ihnen unternommen.

Im Vergleich zu der Anwendung der konventionellen Selbsthilfeeffekt Acrylharze, hat die Einführung Technik mehrere wichtige Vorteile: 1. Eine direkte Verbindung: Selbst Akrylat kann bis auf die Knochen für eine Weile befestigt werden aber nicht bis auf die Knochen halten. Daher sind Verankerungsschrauben erforderlich, um eine Grundplatte fixieren (siehe Abbildung 2a). Im Gegensatz dazu kann Lichthärtung Kunstharzbindung bis auf die Knochen direkt so Schrauben sind nicht erforderlich (Abbildung 2B). Deshalb können G...

Foto 1A zeigt Mikrodialysesonde (a), einer Überwurfmutter (b) und eine Anleitung Kanüle (c). Auf diesem Foto ist die Führung Kanüle durch zahnärztliche Harz, das an den Schädel hält direkt umgeben. Foto B zeigt ein Vogel während der Droge Perfusion. Auf der Oberseite des Kopfes, gibt es eine Mikrodialysesonde, dass zu einer Geldstrafe tefron Rohr verbunden ist. Maßstabsbalken = 1 cm für A-und 2 cm für B sind. <img src="http://www.jove.com/files/249_2008_8_26_3/249_Figure-01.png" /> Abbildung 1 - Fotografien einer Mikrodialyse-Sonde (A) und ein Vogel während der Droge Perfusion (B)

application of light-cured dental adhesive resin for mounting electrodes or microdialysis probes in chronic experiments

Bei der chronischen Aufnahme-Experimente haben selbsthärtenden zahnärztliche Acrylharzen als Grundplatte von Elektroden oder Mikrodialyse-Sonden verwendet worden. Da diese Acrylfarben nicht Bindung an den Knochen haben Schrauben als Anker benutzt worden. Allerdings in kleinen Versuchstieren wie Finken oder Maus, sind ihre Schädel sehr zerbrechlich und kann nicht erfolgreich halten die Anker. In diesem Bericht schlagen wir eine neue Anwendung von lichthärtenden Dentalharze für Montagesockel von Elektroden oder Mikrodialysesonden in chronischen Experimenten. Dieses Material ermöglicht die direkte Anbindung an die Schädeldecke. Daher sind Ankerschrauben nicht erforderlich und chirurgischen Bereich erheblich reduziert werden. Die bisherigen Erfahrungen zeigen, dass die Klebewirkung mehr als 2 Monate hält. Konventionelle Harzes Zeitfenster, wenn die Materialien weich und bearbeitbar sind nur ein paar Minuten. Allerdings ist das Fenster der Arbeitszeit für diese Dentaladhäsive deutlich breiter und einstellbar.

Watch this Scientific Journal Video about Application of Light-cured Dental Adhesive Resin for Mounting Electrodes or Microdialysis Probes in Chronic Experiments at JoVE.com

Application of Light-cured Dental Adhesive Resin for Mounting Electrodes or Microdialysis Probes in Chronic Experiments

In diesem Bericht schlagen wir eine neue Anwendung von lichthärtenden Dentalharze für Montagesockel von Elektroden oder Mikrodialysesonden in chronischen Experimenten. Dieses Material ermöglicht die direkte Anbindung an die Schädeldecke.

Anwendung von Licht-gehärteten Dental Adhesive Resin für Montage-Elektroden oder Mikrodialysesonden in Chronic Experimente

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these ...

application-light-cured-dental-adhesive-resin-for-mounting-electrodes

Research

JoVE Journal

Biology

15.3K Aufrufe.  RIKEN. In diesem Bericht schlagen wir eine neue Anwendung von lichthärtenden Dentalharze für Montagesockel von Elektroden oder Mikrodialysesonden in chronischen Experimenten. Dieses Material ermöglicht die direkte Anbindung an die Schädeldecke.

Serie: Application of Light-cured Dental Adhesive Resin for Mounting Electrodes or Microdialysis Probes in Chronic Experiments

Preparation of Artificial Bilayers for Electrophysiology Experiments

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. These bilayers can be used for many different studies, such as the characterization of membrane-active peptides, the reconstitution of ion channels or investigations on how changes in lipid bilayer properties alter the function of bilayer-spanning channels.  Here, we show how to form a planar bilayer and how to isolate small patches from the bilayer, and in a second video  will also demonstrate a procedure for using gramicidin channels to determine changes in lipid bilayer elastic properties.   We also demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. Here, we demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.

Herstellung künstlicher Doppelschichten für Elektrophysiologie Experimente

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. These bilayers can ...

Forschung

Biologie

Fabrication of Amperometric Electrodes

Carbon fiber electrodes are crucial for the detection of catecholamine release from vesicles in single cells for amperometry measurements.  Here, we describe the techniques needed to generate low noise (&lt;0.5 pA) electrodes.  The techniques have been modified from published descriptions by previous researchers (1,2).  Electrodes are made by preparing carbon fibers and threading them individually into each capillary tube by using a vacuum with a filter to aspirate the fiber.  Next, the capillary tube with fiber is pulled by an electrode puller, creating two halves, each with a fine-pointed tip.  The electrodes are dipped in hot, liquid epoxy mixed with hardener to create an epoxy-glass seal.  Lastly, the electrodes are placed in an oven to cure the epoxy.  Careful handling of the electrodes is critical to ensure that they are made consistently and without damage.  This protocol shows how to fabricate and cut amperometric electrodes for recording from single cells.

This protocol describes how to generate carbon fiber electrodes. The electrodes are subsequently used to detect catecholamine release from vesicles with carbon fiber amperometry.

Herstellung von amperometrische Elektroden

Carbon fiber electrodes are crucial for the detection of catecholamine release from vesicles in single cells for amperometry measurements.  Here, we ...

Robust 3D DNA FISH Using Directly Labeled Probes

3D DNA FISH has become a major tool for analyzing three-dimensional organization of the nucleus, and several variations of the technique have been published. In this article we describe a protocol which has been optimized for robustness, reproducibility, and ease of use. Brightly fluorescent directly labeled probes are generated by nick-translation with amino-allyldUTP followed by chemical coupling of the dye. 3D DNA FISH is performed using a freeze-thaw step for cell permeabilization and a heating step for simultaneous denaturation of probe and nuclear DNA. The protocol is applicable to a range of cell types and a variety of probes (BACs, plasmids, fosmids, or Whole Chromosome Paints) and allows for high-throughput automated imaging. With this method we routinely investigate nuclear localization of up to three chromosomal regions.

We describe a robust and versatile protocol for analyzing nuclear architecture by 3D DNA FISH using directly labeled fluorescent probes.

Robust 3D DNA FISH Mit Direkt markierten Sonden

3D DNA FISH has become a major tool for  analyzing three-dimensional organization of the nucleus, and several variations  of the technique have been ...

Multilayer Mounting for Long-term Light Sheet Microscopy of Zebrafish

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is particularly suited for long-term time-lapse imaging over many hours up to several days. However, an appropriate sample mounting strategy is needed that offers both confinement and normal development of the sample. Multilayer mounting, a new embedding technique using low-concentration agarose in optically clear tubes, now overcomes this limitation and unleashes the full potential of light sheet microscopy for real-time developmental biology.

The development of zebrafish can be followed over days with light sheet microscopy when embryos are embedded in optically clear polymer tubes with low-concentration agarose.

Multilayer-Halterung für Langzeit-Licht-Mikroskopie Blatt der Zebrafisch

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Isolation und Kultur der Dental epithelialen Stammzellen aus dem erwachsenen Maus Schneidezahn

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

The Assembly and Application of &#039;Shear Rings&#039;: A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

Die Montage und Anwendung der &quot;Shear Ringe &#39;: A Novel Endothelial Modell für Orbital, unidirektional und periodische Strömungs- und Scherspannung

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

Application of Genetically Encoded Fluorescent Nitric Oxide (NO&#8226;) Probes, the geNOps, for Real-time Imaging of NO&#8226; Signals in Single Cells

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence of a large number of methods for detecting NO&#8226; in vivo and in vitro, the real-time monitoring of NO&#8226; at the single-cell level is very challenging. The physiological or pathological effects of NO&#8226; are determined by the actual concentration and dwell time of this radical. Accordingly, methods that allow the single-cell detection of NO&#8226; are highly desirable. Recently, we expanded the pallet of NO&#8226; indicators by introducing single fluorescent protein-based genetically encoded nitric oxide (NO&#8226;) probes (geNOps) that directly respond to cellular NO&#8226; fluctuations and, hence, addresses this need. Here we demonstrate the usage of geNOps to assess intracellular NO&#8226; signals in response to two different chemical NO&#8226;-liberating molecules. Our results also confirm that freshly prepared 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine (NOC-7) has a much higher potential to evoke change in intracellular NO&#8226; levels as compared with the inorganic NO&#8226; donor sodium nitroprusside (SNP). Furthermore, dual-color live-cell imaging using the green geNOps (G-geNOp) and the chemical Ca2+ indicator fura-2 was performed to visualize the tight regulation of Ca2+-dependent NO&#8226; formation in single endothelial cells. These representative experiments demonstrate that geNOps are suitable tools to investigate the real-time generation and degradation of single-cell NO&#8226; signals in diverse experimental setups.

Application of Genetically Encoded Fluorescent Nitric Oxide (NO&amp;#8226;) Probes, the geNOps, for Real-time Imaging of NO&amp;#8226; Signals in Single Cells

This manuscript presents protocols for the application of novel genetically encoded nitric oxide (NO&#8226;) probes (geNOps) to monitor single cell NO&#8226; fluctuations in real-time using fluorescence microscopy. The Ca2+-triggered NO&#8226; formation on the level of individual endothelial cells was visualized by combining geNOps with a chemical Ca2+ sensor.

Die Anwendung von genetisch kodierter Fluorescent Stickstoffmonoxid (NO) • Sonden, die geNOps, für Echtzeit-Imaging von NO • Signale in Einzelzellen

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence ...

Inducing Apical Periodontitis in Mice

The mechanisms involved in local induced inflammation can be studied using several available animal models. One of these is the induction of apical periodontitis (AP). Apical periodontitis is a common pathology of an inflammatory nature in the periodontal tissues surrounding the tooth root. In order to better understand the nature and mechanism of this pathology it is advantageous to perform the procedure in mice. The induction of this odontogenic inflammation is achieved by drilling into the mouse tooth until the dental pulp is exposed. Next, the tooth pulp remains exposed to be contaminated by the natural oral flora over time, causing apical periodontitis. After this time period, the animal is sacrificed, and the tooth and the jaw bone can be analyzed in various ways. Typical analyses include micro-CT imaging (to evaluate bone resorption), histological staining, immunohistochemistry, and RNA expression. This protocol is useful for research in the field of oral biology to better understand this inflammatory process in an in vivo experimental setting with uniform conditions. The procedure requires a careful handling of the mice and the isolated jaw, and a visual demonstration of the technique is useful. All technical aspects of the procedures leading to induced apical periodontitis and its characterization in a mouse model are demonstrated.

Here, we present a protocol to locally induce apical periodontitis in mice. We show how to drill a hole in the mouse&#39;s tooth and expose its pulp, in order to cause local inflammation. Analysis methods to investigate the nature of this inflammation, such as micro-CT and histology, are also demonstrated.

Induzieren apische Parodontitis bei Mäusen

The mechanisms involved in local induced inflammation can be studied using several available animal models. One of these is the induction of apical ...

Systematic Assessment of Mammalian Skull Specimens for Dental and Temporomandibular Joint Pathology

Museum skull specimens represent a non-invasive, informative, and readily available means to study temporomandibular joint (TMJ) lesions, dental pathology, and anatomic variations in many mammalian species. Studying the teeth and jaws of an array of species can present a challenge requiring attention to detail and understanding of a species&#39; normal anatomy. In the present article, a systematic and precise protocol for examining skull specimens is discussed that has been applied to a variety of mammals to define characteristic diseases in the oromaxillofacial region. The procedure outlined is simultaneously precise, repeatable, and adaptable to the highly differing skull and tooth shapes and anatomy across species. Specifically, specimens are examined for missing teeth, periodontal disease, endodontal disease, TMJ pathology, and anatomical variations. Results gleaned from research on museum specimens may reflect the natural history, health, and disease status of individuals and species. Furthermore, these data can inform ecological and conservation research efforts, as well as the care of captive individuals.

The present protocol describes the techniques for the systematic assessment of skull specimens to characterize anatomical and developmental variations and abnormalities of the teeth, periodontal disease, endodontal disease, and temporomandibular joint pathology.

Systematische Beurteilung von Schädelpräparaten von Säugetieren auf Zahn- und Kiefergelenkspathologie

Museum skull specimens represent a non-invasive, informative, and readily available means to study temporomandibular joint (TMJ) lesions, dental ...

Etablierung der Primärkultur von Stammzellen der Zahnpulpa

Primary Culture of Dental Pulp Stem Cells

The human dental pulp represents a promising multipotent stem cell reservoir with pre-eminent regenerative competence that can be harvested from an extracted tooth. The neural crest-derived ecto-mesenchymal origin of dental pulp stem cells (DPSCs) bestows a high degree of plasticity that owes to its multifaceted benefits in tissue repair and regeneration. There are various practical ways of harvesting, maintaining, and proliferating adult stem cells being investigated for their use in regenerative medicine. In this work, we demonstrate the establishment of a primary mesenchymal stem cell culture from dental tissue by the explant culture method. The isolated cells were spindle-shaped and adhered to the plastic surface of the culture plate. The phenotypic characterization of these stem cells showed positive expression of the international society of cell therapy (ISCT)-recommended cell surface markers for MSC, such as CD90, CD73, and CD105. Further, negligible expression of hematopoietic (CD45) and endothelial markers (CD34), and less than 2% expression of HLA-DR markers, confirmed the homogeneity and purity of the DPSC cultures. We further illustrated their multipotency based on differentiation to adipogenic, osteogenic, and chondrogenic lineages. We also induced these cells to differentiate into hepatic-like and neuronal-like cells by adding corresponding stimulation media. This optimized protocol will aid in the cultivation of a highly expandable population of mesenchymal stem cells to be utilized in the laboratory or for preclinical studies. Similar protocols can be incorporated into clinical setups for practicing DPSC-based treatments.

Autor im Rampenlicht: Förderung der Geweberegeneration und der Modellierung von Krankheiten mit Stammzellen aus der Zahnpulpa

This article provides a stepwise guide to establish a primary culture of dental pulps stem cells using the explant culture method and characterization of these cells based on ICSCRT guidelines. The cells isolated by this protocol can be considered as mesenchymal stem cells for further applications.

Primärkultur von Stammzellen der Zahnpulpa

The human dental pulp represents a promising multipotent stem cell reservoir with pre-eminent regenerative competence that can be harvested from an ...