Characterizing TMJ Disc Cells: Filament Analysis and Differentiation Role (90 characters), Exams of Anatomy

Download Characterizing TMJ Disc Cells: Filament Analysis and Differentiation Role (90 characters) and more Exams Anatomy in PDF only on Docsity! 1 Semester 1 Examinations 2011/ 2012 Exam Code(s) 4BS2, 4BO2 Exam(s) Fourth year Undenominated Science, Fourth year Biomedical Science Module Code(s) AN435 Module(s) Advanced Anatomy I Paper No. 1 Repeat Paper No External Examiner(s) Professor A. Payne Internal Examiner(s) Dr S.McMahon, Mr A. Black, Professor P. Dockery Instructions: There are two sections, Section A and B. Answer all questions. Duration 3 hours No. of Pages 13 including this coversheet Discipline(s) Anatomy Course Co-ordinator(s) Dr Siobhan McMahon (ext 2838), Mr Alex Black (ext 2234) Requirements: MCQ Release to Library: Yes No Handout Statistical/ Log Tables Cambridge Tables Graph Paper Log Graph Paper Other Materials Please ensure this paper is printed in color PTO 2 OLLSCOIL NA hÉIREANN, GAILLIMH NATIONAL UNIVERSITY OF IRELAND, GALWAY Time Allowed 3 Hours Answer all questions. SECTION A (60% of marks) You have been supplied with the introduction, methods, results and discussion of a research paper 1. Write an abstract for the paper (approximately 300 words) (40 marks) 2. Create a title for the paper (5 marks) 3. What is the aim of the paper? (5 marks) 4. Do the experimental design and the protocols employed control for all potential confounding factors? (5 marks) 5. Are important experimental observations from previous reports described in the context of the present results? (5 marks) SECTION B (40% of marks) Tables / Figures have been provided from a research paper for interpretation 6. Describe the results and outline what conclusions can be drawn from the data shown in Figure 1 (20 marks) 7. Describe the results and outline what conclusions can be drawn from the data shown in Figure 2 (20 marks) 5 overnight at room temperature. They were incubated with a biotinylated horse anti-mouse IgG (1 : 100; Vector Lab., Burlingame, CA, USA), followed with horseradish peroxidase- conjugated avidin (ABC kit, Vector Lab.). Final visualization used 3, 3′-diaminobenzidine (0.04%) and hydrogen peroxide (0.003%) in a 0.05 m Tris-HCl buffer at pH 7.6. The immunostained sections were counterstained with 0.03% methylene blue. For immunocytochemistry at the electron microscopic level, we used a nanogold-conjugated mouse IgG (Nanoprobes, Yaphank, NY, USA) – instead of a biotinylated one – and a silver enhancement technique for cryostat sections. These immunoreacted sections were osmificated, dehydrated through ascending ethanols, and finally embedded in an epoxy resin (Epon 812; TAAB, Berkshire, UK). Ultrathin sections, 70 nm thick, were prepared with an ultramicrotome (Ultracut R, Leica) and briefly double-stained with uranyl acetate and lead citrate. They were examined in an H-7650 transmission electron microscope at an accelerating voltage of 75 kv (Hitachi, Tokyo, Japan). Double-fluorescent immunostaining A double-immunolabeling was performed in cryostat sections for the identification of nestin- or GFAP-immunoreactive cells. In addition, a co-localization of Hsp25, a marker for the articular disc cells, and either nestin or GFAP was also checked by a double- immunofluorescent method. Information on the primary and secondary antibodies used in this study is shown in Table 1. The nucleus was stained with DAPI (Vector Lab.). The immunostained sections were observed with a fluorescent microscope (AxioImazer M; Carl Zeiss, Jena, Germany). Table 1. Antibodies used for double immunostaining. Monoclonal Fluorescent dye Polyclonal Fluorescent dye Nestin (clone: Rat- 401) 1 : 2500, Chemicon International, Temecula, CA, USA Cy3 1 : 500, Jackson ImmunoResearch Lab., West Grove, PA, USA GFAP 1 : 4000, Dako, Glostrup, Denmark FITC 1 : 200, Vector Lab., Burlingame, CA, USA GFAP (clone:GA5) 1 : 2000, Millipore, Temecula, CA, USA Hsp 25 (poly) 1 : 1000, Stressgen Biotechnologies, Victoria, Canada 6 Figure 1. Immunoreactions for nestin (A, B) and GFAP (C, D) at postnatal day 1. (A, B) A few slender cells with nestin expression (arrow in A) are observed along the anterior portion of the articular disc (D) surface. The central portion of the disc never contains any distinct positive cells (B). However, the invading blood capillaries into the future lower articular cavity area show intense nestin immunoreaction (arrows in B). (C, D) Some cells with strong GFAP immunoreaction exist in the deeper area of the anterior articular disc (arrows), although immunoreaction for GFAP exists neither in the central area of the disc (D) nor blood capillaries (BV) between the surface of the mandibular condyle and the disc. Scale bars: 20 μm. T, temporal bone. Results  Postnatal day 1 At this stage, the lower articular cavity had not yet completely formed, although the upper articular cavity had expanded in the anterior–posterior direction. As we reported previously (Suzuki et al. 2005), the articular disc was not separated from the condylar surface where several blood capillaries lay at the central portion of the lower articular cavity. A very few cells immunoreacting with nestin existed at the anterior and posterior portions of the articular disc (Fig. 1A). Flat in profile, they were located near the articular disc surface but never in the deep area of the articular disc. The nestin-positive cells sometimes extended their thin cytoplasmic extensions towards the articular cavity (arrow in Fig. 1A). The immunoreaction for nestin was localized in the cytoplasm devoid of the nucleus. In contrast, our careful observations could find no nestin-positive cells at the central portion of the articular disc (Fig. 1B). The endothelial cells between the articular disc and condylar surface also expressed nestin immunopositivity (Fig. 1A,B). On the other hand, an expression of GFAP immunoreaction was recognizable in a few cells with poor cytoplasm situated in the deep areas of the anterior and posterior portions (Fig. 1C), but not in the central portions (Fig. 1D). However, the endothelial cells lacked any GFAP immunoreactivity. Postnatal week 1 At postnatal week 1, formation of articular cavity had completely finished and the area of the articular disc was clearly divided into three portions: a thick anterior band, a thin intermediate zone, and a thick posterior band – all of which were observed in mature animals. Compared with the previous stage, the nestin- or GFAP-immunoreactive cells had increased in number (Fig. 2). The location and morphology of the nestin-positive cells were the same as those of the reactive cells at the previous stage: at the anterior and posterior bands they existed near the disc surface and possessed thin cytoplasmic extensions (Fig. 2A). At the intermediate zone, slender cells with nestin immunopositivity came to arrange themselves 7 Figure 2. Immunoreactions for nestin (A, B) and GFAP (C, D) in the anterior (A, C) and central portions (B, D) of the developing articular disc at postnatal week 1. (A) Some nestin-positive cells with thin cytoplasmic projections (arrows) appear near the surface at the anterior portion. (B) The center of the articular disc (D) comes to contain some slender cells expressing nestin immunoreaction at the surface of the disc (arrows). (C, D) Almost all cells in the articular disc have an immunopositive reaction for GFAP. Some GFAP-positive cells exhibit a strong GFAP immunoreaction (arrows). Scale bars: 20 μm. C, mandibular condyle; T, temporal bone. Figure 3. Immunoexpression of nestin (A–C) and GFAP (D–F) in the articular disc (D) at postnatal week 2. (A–C) The nestin-positive cells extend their thin cytoplasmic processes (arrows) in the anterior band (A) and intermediate zone (B) of the disc. An arrowhead indicates a nestin-positive cell at the surface (B). In the posterior band (C), endothelial cells (arrowheads) are positive in nestin immunoreaction. (D–F) The central area of the disc contains a major population of disc cells with strong GFAP immunoreactions, while the peripheral area has weakly positive cells (D). These strong positive cells assume large round profiles with long and thick cytoplasmic projections in the anterior portion (E) or flat profiles with thick ramified processes in the intermediate zone (F). Scale bars: 20 μm in (A–C, E, F), 100 μm in (D). C, mandibular condyle; T, temporal bone. along the disc surface (Fig. 2B). Some cells with poor cytoplasm also exhibited nestin immunoreaction in deep areas (Fig. 2A). Many articular disc cells enlarged to have GFAP immunoreaction, though GFAP immuno- intensity varied among the positive cells (Fig. 2C,D). However, there was no GFAP-positive cell that lined the articular cavity, unlike the nestin-positive cells. Postnatal week 2 The articular disc at this stage had become obviously thicker in anterior and posterior bands than at the previous stage. In addition to the articular surface, nestin-positive cells appeared in the deep area of the articular disc (Fig. 3A,B). They extended thin cytoplasmic processes. The endothelial cells in the deep area were also positive in nestin immunoreaction (Fig. 3C). 10 The articular disc cells with nestin immunoreaction were characterized by many short and thin cytoplasmic processes – appearing as pseudopodia-like processes – and an indented nucleus with clear chromatin (Fig. 6A). Their poor cytoplasm contained a few cell organellae, including a rough-surfaced endoplasmic reticulum and Golgi complex (Fig. 6B). Some large mitochondria were scattered in their cytoplasm. The nestin immunoreaction was recognizable on comparatively thin and short intermediate filaments, which did not form fiber bundles (Fig. 6C). The GFAP-reactive cells featured a large, clear, irregular nucleus and thick and long cell process (Fig. 6D). They had also developed cell organellae such as a rough-surfaced endoplasmic reticulum and Golgi complex (Fig. 6E) in their cytoplasm and caveola-like invaginations along their cell membranes (Fig. 6E). The GFAP-reactive intermediate filaments formed thick bundles that appeared to surround the nucleus (Fig. 6F). The phenotypes of the articular disc cells in mature rats are summarized in Table 2. Table 2. Immunological and morphological characterizations of the rat articular disc. Hsp25 Co-localization of immunoreaction Feature Nestin GFAP + − + Large nucleus and rich cytoplasm + − + Thick and long cell processes + − + A major population of articular disc cells + − − Present near the articular surface − + − Small nucleus and poor cytoplasm − + − Thin and short cell processes − + − Scattered in the articular disc   Discussion  The temporomandibular joint, which plays crucial roles in complicated jaw movements, is a bilateral symmetric synovial joint between the head of the mandibular condyle and the mandibular fossa of the temporal bone (Bernick, 1987). The articular disc, a major component of the temporomandibular joint, is usually exposed to various mechanical stresses, including intermittent occlusal force. Histological observations demonstrate that this structure is an avascular fibrous tissue consisting of a dense network of collagen fibers and disc cells called chondrocyte-like cells or fibrochondrocytes (Berkovitz & Pacy, 2000, 2002). The location and structure of the articular disc readily suggest that the articular disc cells serve in its maintenance and preservation. From the viewpoint of the expression patterns of intermediate filaments and Hsp25, the present immunohistochemical study was able to reveal at least three phenotypes in the rat articular disc: articular disc cells with Hsp25 (+)/nestin (−)/GFAP (+); with Hsp25 (+)/nestin (−)/GFAP (−); and with Hsp25 (−)/nestin (+)/GFAP (−) (Table 2). Because a previous study failed to find Hsp25-negative cells in the articular disc (Nozawa- Inoue et al. 1999), this is the first report to demonstrate another type of articular disc cell that is devoid of Hsp25 immunoreaction. In immunoelectron microscopic observations, the Hsp25 (−)/nestin (+)/GFAP (−) disc cells were characteristic of a poor cytoplasm with fewer cell organellae – a feature common to the nestin-positive disc cells, suggesting that this is a kind 11 of immature or undifferentiating cell. There is no evidence of whether this immature type of cell can differentiate into GFAP- and/or Hsp25-positive cells with thick cell processes and a rich cytoplasm, though nestin is replaced by GFAP in nervous tissue during development (Lendahl et al. 1990). It is notable that a smaller number of articular disc cells showed immunoreaction for nestin. This protein was first discovered in rat neuroepithelial stem cells (Hockfield & Mckay, 1985; Lendahl et al. 1990), but recent studies have confirmed its expression in many non-neuronal cells (cf. Wiese et al. 2004). Current immunohistochemical observations at light and electron microscopic levels indicate that the articular disc cells with nestin immunoreaction may have a capacity for differentiation in the mature articular disc, as shown in the human knee meniscus, which has been reported to contain mesenchymal stem cells (Segawa et al. 2009). Although our preliminary study has shown a low level of apoptosis and mitosis in the articular disc cells (unpublished data), the existence of nestin-expressing cells implies the possibility that they have an ability to differentiate into the other types of cells – including endothelial cells and pericytes – for repair of the damaged articular disc. It is well known that temporomandibular joint plays crucial roles in jaw movement. Occlusion between upper and lower teeth is an important factor that regulates jaw movement (Matthews, 1975). Previous studies have suggested the involvement of occlusal forces in the development of the extracellular matrices in the articular disc of the temporomandibular joint (Toriya et al. 2006; Feng et al. 2010). These findings readily prompted us to hypothesize a close relationship between mechanical stress and the development of the intermediate filaments in the articular disc. In this study, we divided the observation period into five stages. This classification is based on the status of tooth eruption and occlusion (Nakakura-Ohshima et al. 1993, 1995): postnatal day 1 (incisors not erupted) and postnatal weeks 1 (upper and lower incisors erupted and the occlusion of the incisors begun), 2 (the commencement of occlusion having started between upper and lower molars), and 4 and 8 (occlusion established between upper and lower molars). Current observations of the rat temporomandibular joint showed that the immunoexpression patterns of nestin and GFAP in the articular disc cells reflected the developmental process of the articular disc: no remarkable changes in the immunoexpression pattern, and the morphology and distribution of immunoreactive articular disc cells was recognizable after the occlusion of the upper and lower molars was established. This finding indicates that the addition of mechanical stress via teeth may contribute to the maturation of the articular disc. In particular, as the GFAP-expressing cells drastically increased in number when occlusion had commenced between upper and lower molars, it is possible that the GFAP immunoexpression is a key indicator of the maturation of the articular disc. In conclusion, the articular disc cells comprise at least three types in the rat temporomandibular joint as defined by the expression patterns of intermediate filaments, including nestin and GFAP. This study was, however, carried out on intact rats through their postnatal development. Further observations on older animals and under pathological conditions including over-loading or loss of occlusal force are needed for clarifying the significance of the expression of these intermediate filaments. 12 SECTION B The following Figures have been provided from research papers for interpretation Figure 1. Decorin, fibronectin, and collagen I dose optimization. A) decorin dose response at 5, 10 and 20 μg/ml concentrations. B) fibronectin dose response at 5, 10 and 20 μg/ml concentrations. C) collagen I at 6.25, 12.5, 25, 50, and 100 μg/ml concentrations. A scratch assay was utilized with photos taken at hours 0, 1, 3, 5, and 7. Growth media on non-coated wells was used as control for all experimental procedures. Percentage wound closure was calculated by determining the area of the wound. *p < 0.05, **p < 0.005, n = 3. All data shown as ± standard error of the mean (SEM)