Faculty of Dentistry

Permanent URI for this communityhttps://hdl.handle.net/1807/16909

Internationally known for its academic achievements in both clinical and research, the Faculty of Dentistry at the University of Toronto is the champion of dental school in Canada. Since founded in 1875 by the Royal College of Dental Surgeons of Ontario, the Faculty has continuously educated top-notch specialists in Orthodontics, Paediatric, Dentistry, Periodontology, Endodontics, Prosthodontics, and many more disciplines. One of the major commitments of the faculty is to dental research as no such research is carried out by private industry or government bodies. On the same page, the faculty also takes on a multi-disciplinary approach as it participates in a number of inter-faculty programs.

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Time scale and other invariants of integrative mechanical behavior in living cells
(American Physical Society, 2003) Fabry, B.; Maksym, G. N.; Butler, J. P.; Glogauer, Michael; Navajas, D.; Taback, N. A.; Millet, E. J.; Fredberg, J. J.
The role of TLR4 and neutrophil expression in infection-triggered arthritis
(Biomed Central, 2004) Zhang, X.; Glogauer, Michael; Zhu, F.; Kim, T.; Chiu, B.; Inman, RD
The N. gonorrhoeae type IV pilus stimulates mechanosensitive pathways and cytoprotection through a pilT-dependent mechanism
(Public Library of Science, 2005) Howie, H. L.; Glogauer, Michael; So, M.
Real-Time High Resolution 3D Imaging of the Lyme Disease Spirochete Adhering to and Escaping from the Vasculature of a Living Host
(Public Library of Science (PLoS), 2008-06-20) Moriarty, Tara ; Norman, M. Ursula ; Colarusso, Pina ; Bankhead, Troy ; Kubes, Paul ; Chaconas, George
Pathogenic spirochetes are bacteria that cause a number of emerging and re-emerging diseases worldwide, including syphilis, leptospirosis, relapsing fever, and Lyme disease. They exhibit an unusual form of motility and can infect many different tissues; however, the mechanism by which they disseminate from the blood to target sites is unknown. Direct visualization of bacterial pathogens at the single cell level in living hosts is an important goal of microbiology, since this approach is likely to yield critical insight into disease processes. We engineered a fluorescent strain of Borrelia burgdorferi, a Lyme disease pathogen, and used conventional and spinning disk confocal intravital microscopy to directly visualize these bacteria in real time and 3D in living mice. We found that spirochete interaction with and dissemination out of the vasculature was a multi-stage process of unexpected complexity and that spirochete movement appeared to play an integral role in dissemination. To our knowledge, this is the first report of high resolution 3D visualization of dissemination of a bacterial pathogen in a living mammalian host, and provides the first direct insight into spirochete dissemination in vivo.
Healing of periodontal tissues following transplantation of cells in a rat orthodontic tooth movement model
(Edward H. Angle Society of Orthodontists, 2008-09) Nayak, B. N.; Wiltshire, W. A.; Ganss, B.; Tenenbaum, H.; McCulloch, C. H.; Lekic, C.
OBJECTIVE: To determine the fate and differentiation of transplanted periodontal ligament (PL) precursor cells and mouse embryonic stem (ES) cells and their relative capacity to regenerate wounded periodontium. MATERIALS AND METHODS: Orthodontic tooth movement was introduced 24 hours before transplantation of PL or ES cells, and rats were euthanized either 24 hours or 72 hours after cell transplantation. The control rats received either no tooth movement and no cell transplantation or tooth movement and no cell transplantation. Differentiation of transplanted cells was assessed from mandibular periodontal histological tissue sections by immunohistochemical methods using monoclonal antibodies against PL cell differentiation markers. Data were analyzed using Student's t-test at a significance level of P = .05. RESULTS: Transplantation of PL and ES cells resulted in a higher number of osteopontin, bone sialoprotein, and alpha-smooth muscle actin labeled transplanted cells, predominantly around the blood vessels of the periodontium in study rats compared with control rats (cell transplantation but no orthodontic tooth movement, P = .05). Combined treatments of tooth movement and cell transplantation resulted in enhanced regeneration of the periodontium as a result of tooth movement. Transplantation of PL cells induced a higher number of differentiating cells in the PL and alveolar bone than did transplantation of ES cells. CONCLUSIONS: Orthodontic tooth movement promotes the differentiation of transplanted cells, and the differentiation occurs predominantly in the paravascular areas of the periodontium. In terms of regeneration of wounded periodontium, transplantation of PL cells produced a higher level of regeneration than ES cells, possibly because of PL cell plasticity and the capacity to undergo effective differentiation in the periodontal cellular microenvironment.
Rap1 Activation in Collagen Phagocytosis Is Dependent on Nonmuscle Myosin II-A
(American Society for Cell Biology, 2008-12) Arora, Pamela D. ; Conti, Mary Anne ; Ravid, Shoshana ; Sacks, David B. ; Kapus, Andras ; Adelstein, Robert S. ; Bresnick, Anne R. ; McCulloch, Christopher A.
Rap1 enhances integrin-mediated adhesion but the link between Rap1 activation and integrin function in collagen phagocytosis is not defined. Mass spectrometry of Rap1 immunoprecipitates showed that the association of Rap1 with nonmuscle myosin heavy-chain II-A (NMHC II-A) was enhanced by cell attachment to collagen beads. Rap1 colocalized with NM II-A at collagen bead-binding sites. There was a transient increase in myosin light-chain phosphorylation after collagen-bead binding that was dependent on myosin light-chain kinase but not Rho kinase. Inhibition of myosin light-chain phosphorylation, but not myosin II-A motor activity inhibited collagen-bead binding and Rap activation. In vitro binding assays demonstrated binding of Rap1A to filamentous myosin rods, and in situ staining of permeabilized cells showed that NM II-A filaments colocalized with F-actin at collagen bead sites. Knockdown of NM II-A did not affect talin, actin, or β1-integrin targeting to collagen beads but targeting of Rap1 and vinculin to collagen was inhibited. Conversely, knockdown of Rap1 did not affect localization of NM II-A to beads. We conclude that MLC phosphorylation in response to initial collagen-bead binding promotes NM II-A filament assembly; binding of Rap1 to myosin filaments enables Rap1-dependent integrin activation and enhanced collagen phagocytosis.
The Role of FilGAP-Filamin A Interactions in Mechanoprotection
(American Society for Cell Biology, 2009-03-01) Shifrin, Yulia ; Arora, Pamela D. ; Ohta, Yasutaka ; Calderwood, David A. ; McCulloch, Christopher A.
Cells in mechanically active environments are subjected to high-amplitude exogenous forces that can lead to cell death. Filamin A (FLNa) may protect cells from mechanically induced death by mechanisms that are not yet defined. We found that mechanical forces applied through integrins enhanced Rac-mediated lamellae formation in FLNa-null but not FLNa-expressing cells. Suppression of force-induced lamella formation was mediated by repeat 23 of FLNa, which also binds FilGAP, a recently discovered Rac GTPase-activating protein (GAP). We found that FilGAP is targeted to sites of force transfer by FLNa. This force-induced redistribution of FilGAP was essential for the suppression of Rac activity and lamellae formation in cells treated with tensile forces. Depletion of FilGAP by small interfering RNA, inhibition of FilGAP activity by dominant-negative mutation or deletion of its FLNa-binding domain, all resulted in a dramatic force-induced increase of the percentage of annexin-V-positive cells. FilGAP therefore plays a role in protecting cells against force-induced apoptosis, and this function is mediated by FLNa.
Activation of antibacterial autophagy by NADPH oxidases
(National Academy of Sciences, 2009-04-14) Huanga, Ju; Canadiena, Veronica; Lama, Grace Y.; Steinberga, Benjamin E.; Dinauer, Mary C.; Magalhaes, Marco A. O.; Glogauer, Michael; Grinstein, Sergio
Autophagy plays an important role in immunity to microbial pathogens. The autophagy system can target bacteria in phagosomes, promoting phagosome maturation and preventing pathogen escape into the cytosol. Recently, Toll-like receptor (TLR) signaling from phagosomes was found to initiate their targeting by the autophagy system, but the mechanism by which TLR signaling activates autophagy is unclear. Here we show that autophagy targeting of phagosomes is not exclusive to those containing TLR ligands. Engagement of either TLRs or the Fcγ receptors (FcγRs) during phagocytosis induced recruitment of the autophagy protein LC3 to phagosomes with similar kinetics. Both receptors are known to activate the NOX2 NADPH oxidase, which plays a central role in microbial killing by phagocytes through the generation of reactive oxygen species (ROS). We found that NOX2-generated ROS are necessary for LC3 recruitment to phagosomes. Antibacterial autophagy in human epithelial cells, which do not express NOX2, was also dependent on ROS generation. These data reveal a coupling of oxidative and nonoxidative killing activities of the NOX2 NADPH oxidase in phagocytes through autophagy. Furthermore, our results suggest a general role for members of the NOX family in regulating autophagy.
Bone sialoprotein does not interact with pro-gelatinase A (MMP-2) or mediate MMP-2 activation
(BioMed Central, 2009-04-22) Hwang, Queena ; Cheifetz, Sela ; Overall, Christopher M. ; McCulloch, Christopher A. ; Sodek, Jaro
Background A recent model for activation of the zymogen form of matrix metalloproteinase 2 (MMP-2, also known as gelatinase A) has suggested that interactions between the SIBLING protein bone sialoprotein (BSP) and MMP-2 leads to conformational change in MMP-2 that initiates the conversion of the pro-enzyme into a catalytically active form. This model is particularly relevant to cancer cell metastasis to bone since BSP, bound to the αvβ3 integrin through its arginine-glycine-aspartic acid motif, could recruit MMP-2 to the cell surface. Methods We critically assessed the relationship between BSP and proMMP-2 and its activation using various forms of recombinant and purified BSP and MMP-2. Gelatinase and collagenase assays, fluorescence binding assays, real-time PCR, cell culture and pull-down assays were employed to test the model. Results Studies with a fluorogenic substrate for MMP-2 showed no activation of proMMP-2 by BSP. Binding and pull-down assays demonstrated no interaction between MMP-2 and BSP. While BSP-mediated invasiveness has been shown to depend on its integrin-binding RGD sequence, analysis of proMMP-2 activation and the level of membrane type 1 (MT1)-MMP in cells grown on a BSP substratum showed that the BSP-αvβ3 integrin interaction does not induce the expression of MT1-MMP. Conclusion These studies do not support a role for BSP in promoting metastasis through interactions with pro-MMP-2.
The axonal repellent, Slit2, inhibits directional migration of circulating neutrophils.
(Society for Leukocyte Biology, 2009-12) Tole, S ; Mukovozov, IM ; Huang, YW ; Magalhaes, MA ; Yan, M ; Crow, MR ; Liu, GW ; Sun, CX ; Durocher, Y ; Glogauer, Michael ; Robinson, Lisa A
In inflammatory diseases circulating neutrophils are recruited to sites of injury. Attractant signals are provided by many different chemotactic molecules, such that blockade of one may not effectively prevent neutrophil recruitment. The Slit family of secreted proteins, and their transmembrane receptor, Roundabout (Robo), repel axonal migration during central nervous system development. Emerging evidence shows that by inhibiting the activation of Rho-family GTPases, Slit2/Robo also inhibit migration of other cell types towards a variety of chemotactic factors, in vitro and in vivo. The role of Slit2 in inflammation, however, has been largely unexplored. We isolated primary neutrophils from human peripheral blood and mouse bone marrow, and detected Robo-1 expression. Using video-microscopic live cell tracking, we found that Slit2 selectively impaired directional migration, but not random movement, of neutrophils towards formyl-methionyl-leucyl-phenylalanine (fMLP). Slit2 also inhibited neutrophil migration towards other chemoattractants, namely C5a and interleukin (IL)-8. Slit2 inhibited neutrophil chemotaxis by preventing chemoattractant-induced actin barbed end formation and cell polarization. Slit2 mediated these effects by suppressing inducible activation of Cdc42 and Rac2, but did not impair activation of other major kinase pathways involved in neutrophil migration. We further tested the effects of Slit2 in vivo using mouse models of peritoneal inflammation induced by sodium periodate, C5a, and macrophage inflammatory protein-2 (MIP-2). In all instances, Slit2 effectively reduced neutrophil recruitment (p < 0.01). Collectively, these data demonstrate that Slit2 potently inhibits chemotaxis, but not random motion, of circulating neutrophils, and point to Slit2 as a potential new therapeutic for preventing localized inflammation.
An intravascular immune response to Borrelia burgdorferi involves Kupffer cells and iNKT cells
(Nature Publishing Group, 2010-04) Lee, W. Y.; Moriarty, T. J.; Wong, C. H.; Zhou, H.; Strieter, R. M.; van Rooijen, N.; Chaconas, G.; Kubes, P.
Here we investigate the dynamics of the hepatic intravascular immune response to a pathogen relevant to invariant natural killer T cells (iNKT cells). Immobilized Kupffer cells with highly ramified extended processes into multiple sinusoids could effectively capture blood-borne, disseminating Borrelia burgdorferi, creating a highly efficient surveillance and filtering system. After ingesting B. burgdorferi, Kupffer cells induced chemokine receptor CXCR3-dependent clustering of iNKT cells. Kupffer cells and iNKT cells formed stable contacts via the antigen-presenting molecule CD1d, which led to iNKT cell activation. An absence of iNKT cells caused B. burgdorferi to leave the blood and enter the joints more effectively. B. burgdorferi that escaped Kupffer cells entered the liver parenchyma and survived despite Ito cell responses. Kupffer cell-iNKT cell interactions induced a key intravascular immune response that diminished the dissemination of B. burgdorferi.
Pivotal Advance: Phospholipids determine net membrane surface charge resulting in differential localization of active Rac1 and Rac2.
(Society for Leukocyte Biology, 2010-04) Magalhaes, MA ; Glogauer, Michael
In this investigation, we used primary murine neutrophils to demonstrate that local changes in membrane phospholipid composition alter the net cytoplasmic membrane surface charge, which results in selective recruitment of Rac1 or Rac2 based on the net charge of their respective C-terminal domains. Murine neutrophils undergoing chemotaxis or carrying out phagocytosis were transfected with K-ras4B-derived membrane charge biosensors and lipid markers, which allowed us to simultaneously monitor the levels of PIP2, PIP3, and PS and net membrane charge of the newly developing phagosome membrane and plasma membrane. Our results indicate that the combination of PIP2, PIP3, and PS generates a high negative charge (–8) at the plasma membrane of actin-rich pseudopods, where active Rac1 preferentially localizes during phagosome formation. The lipid metabolism that occurs during phagosome maturation results in the localized depletion of PIP2, PIP3, and partial decrease in PS. This creates a moderately negative net charge that correlates with the localization of active Rac2. Conversely, the accumulation of PIP3 at the leading-edge membrane during chemotaxis generates a polarized accumulation of negative charges that recruits Rac1. These results provide evidence that alterations in membrane lipid composition and inner-membrane surface charge are important elements for the recruitment of differentially charged proteins and localization of signaling pathways during phagocytosis and chemotaxis in neutrophils.
Isoform-Specific Upregulation of Palladin in Human and Murine Pancreas Tumors
(Public Library of Science (PLoS), 2010-04-26) Goicoechea, Silvia M. ; Bednarski, Brian ; Stack, Christianna ; Cowan, David W. ; Volmar, Keith ; Thorne, Leigh ; Cukierman, Edna ; Rustgi, Anil K. ; Brentnall, Teresa ; Hwang, Rosa F. ; McCulloch, Christopher A. ; Yeh, Jen Jen ; Bentrem, David J. ; Hochwald, Steven N. ; Hingorani, Sunil R. ; Kim, Hong Jin ; Otey, Carol A.
Pancreatic ductal adenocarcinoma (PDA) is a lethal disease with a characteristic pattern of early metastasis, which is driving a search for biomarkers that can be used to detect the cancer at an early stage. Recently, the actin-associated protein palladin was identified as a candidate biomarker when it was shown that palladin is mutated in a rare inherited form of PDA, and overexpressed in many sporadic pancreas tumors and premalignant precursors. In this study, we analyzed the expression of palladin isoforms in murine and human PDA and explored palladin's potential use in diagnosing PDA. We performed immunohistochemistry and immunoblot analyses on patient samples and tumor-derived cells using an isoform-selective monoclonal antibody and a pan-palladin polyclonal antibody. Immunoblot and real-time quantitative reverse transcription-PCR were used to quantify palladin mRNA levels in human samples. We show that there are two major palladin isoforms expressed in pancreas: 65 and 85–90 kDa. The 65 kDa isoform is expressed in both normal and neoplastic ductal epithelial cells. The 85–90 kDa palladin isoform is highly overexpressed in tumor-associated fibroblasts (TAFs) in both primary and metastatic tumors compared to normal pancreas, in samples obtained from either human patients or genetically engineered mice. In tumor-derived cultured cells, expression of palladin isoforms follows cell-type specific patterns, with the 85–90 kDa isoform in TAFs, and the 65 kDa isoform predominating in normal and neoplastic epithelial cells. These results suggest that upregulation of 85–90 kDa palladin isoform may play a role in the establishment of the TAF phenotype, and thus in the formation of a desmoplastic tumor microenvironment. Thus, palladin may have a potential use in the early diagnosis of PDA and may have much broader significance in understanding metastatic behavior.
Tumor Cell Invasion Is Promoted by Interstitial Flow-Induced Matrix Priming by Stromal Fibroblasts.
(American Association for Cancer Research, 2011-02) Shieh, Adrian C. ; Rozansky, Hallie A. ; Hinz, Boris ; Swartz, Melody A.
Interstitial flow emanates from tumors into the microenvironment where it promotes tumor cell invasion. Fibroblasts are key constituents of the tumor stroma which modulate the mechanical environment by matrix remodeling and contraction. Here we explore how interstitial fluid flow affects fibroblast-tumor cell interactions. Using a 3-D invasion assay and MDA-MB-435S cells co-cultured with dermal fibroblasts in a collagen matrix, we demonstrated a synergistic enhancement of tumor cell invasion by fibroblasts in the presence of interstitial flow. Interstitial flow also drove transforming growth factor (TGF)-β1 and collagenase-dependent fibroblast migration, consistent with previously described mechanisms where flow promotes invasion through autologous chemotaxis and increased motility. Concurrently, migrating fibroblasts enhanced tumor cell invasion by matrix priming via Rho-mediated contraction. We propose a model where interstitial flow promotes fibroblast migration through TGF-β1 and collagenase activity, positioning fibroblasts to locally reorganize collagen fibers via Rho-dependent contractility, enhancing tumor cell invasion via mechanotactic cues. This represents a novel mechanism where interstitial flow causes stromal remodeling that facilitates tumor invasion.
Rac1 Deletion Causes Thymic Atrophy
(Public Library of Science (PLoS), 2011-04-29) Hunziker, Lukas ; Benitah, Salvador Aznar ; Jensen, Kim ; McNulty, Katrina ; Butler, Colin ; Potton, Elspeth ; Nye, Emma ; Boyd, Richard ; Laurent, Geoff ; Glogauer, Michael ; Wright, Nick A. ; Watt, Fiona M. ; Janes, Sam M.
The thymic stroma supports T lymphocyte development and consists of an epithelium maintained by thymic epithelial progenitors. The molecular pathways that govern epithelial homeostasis are poorly understood. Here we demonstrate that deletion of Rac1 in Keratin 5/Keratin 14 expressing embryonic and adult thymic epithelial cells leads to loss of the thymic epithelial compartment. Rac1 deletion led to an increase in c-Myc expression and a generalized increase in apoptosis associated with a decrease in thymic epithelial proliferation. Our results suggest Rac1 maintains the epithelial population, and equilibrium between Rac1 and c-Myc may control proliferation, apoptosis and maturation of the thymic epithelial compartment. Understanding thymic epithelial maintenance is a step toward the dual goals of in vitro thymic epithelial cell culture and T cell differentiation, and the clinical repair of thymic damage from graft-versus-host-disease, chemotherapy or irradiation.
Treponema denticola Major Outer Sheath Protein Induces Actin Assembly at Free Barbed Ends by a PIP2- Dependent Uncapping Mechanism in Fibroblasts
(Public Library of Science (PLoS), 2011-08-25) Visser, Michelle B. ; Koh, Adeline ; Glogauer, Michael ; Ellen, Richard P.
The major outer sheath protein (Msp) of Treponema denticola perturbs actin dynamics in fibroblasts by inducing actin reorganization, including subcortical actin filament assembly, leading to defective calcium flux, diminished integrin engagement of collagen, and retarded cell migration. Yet, its mechanisms of action are unknown. We challenged Rat-2 fibroblasts with enriched native Msp. Msp activated the small GTPases Rac1, RhoA and Ras, but not Cdc42, yet only Rac1 localized to areas of actin rearrangement. We used Rac1 dominant negative transfection and chemical inhibition of phosphatidylinositol-3 kinase (PI3K) to show that even though Rac1 activation was PI3K-dependent, neither was required for Msp-induced actin rearrangement. Actin free barbed end formation (FBE) by Msp was also PI3K-independent. Immunoblotting experiments showed that gelsolin and CapZ were released from actin filaments, whereas cofilin remained in an inactive state. Msp induced phosphatidylinositol (4,5)-bisphosphate (PIP2) formation through activation of a phosphoinositide 3-phosphatase and its recruitment to areas of actin assembly at the plasma membrane. Using a PIP2 binding peptide or lipid phosphatase inhibitor, PIP2 was shown to be required for Msp-mediated actin uncapping and FBE formation. Evidently, Msp induces actin assembly in fibroblasts by production and recruitment of PIP2 and release of the capping proteins CapZ and gelsolin from actin barbed ends.
Rac2 is required for the formation of neutrophil extracellular traps.
(Society for Leukocyte Biology, 2011-10) Lim, MB ; Kuiper, JB ; Katchky, A ; Goldberg, H ; Glogauer, Michael
Neutrophils play a critical role as a first line of defence against invading pathogens. Recently, a new defence strategy of neutrophils was described in which pathogens are trapped and killed by neutrophil extracellular traps (NETs). However, the exact underlying mechanisms leading to the formation of NETs remain elusive. Here, we explored the role of Rac small GTPases in the formation of NETs using neutrophils that lack Rac1, Rac2 or both isoforms. Efficient NET formation was observed in both wild-type and Rac1null neutrophils. In contrast, NET formation was markedly impaired in cells lacking either Rac2 or both Rac2 and Rac1. The defect in NET formations in Rac2null cells could be rescued by exogenous ROS sources suggesting that Rac2 mediated ROS generation is required for NET formation. In addition, we assessed the role of nitric oxide in NET formation in murine neutrophils. Blocking nitric oxide (NO) production with the nitric oxide synthase inhibitor, L-NAME significantly reduced NET formation. Moreover, we show that Rac2null cells produce significantly less NO than Rac1null cells or their wildtype counterparts. Our data suggest that Rac2 is essential for NET formation via pathways involving both ROS and NO.
Aquaporin 9 phosphorylation mediates membrane localization and neutrophil polarization.
(Society for Leukocyte Biology, 2011-11) Karlsson, T ; Glogauer, Michael ; Ellen, EP ; Loitto, VM ; Magnusson, KE ; es, MA Magalhã
Neutrophils are of prime importance in the host innate defence against invading microorganisms by employing two primary mechanisms, locomotion towards and phagocytosis of the prey. Recent research points to pivotal roles for water channels known as aquaporins in cell motility. Here, we focused on the role of aquaporin 9 (AQP9) in chemoattractant-induced polarization and migration of primary mouse neutrophils and neutrophil-like HL60 cells. We found that AQP9 is phosphorylated downstream of the fMLF receptor or PMA stimulation in primary human neutrophils. The dynamics of AQP9 was assessed using GFP-tagged AQP9 constructs and other fluorescent markers through various live-cell imaging techniques. Expression of wild-type or the phosphomimic S11D AQP9 changed cell volume regulation as a response to hyperosmotic changes and enhanced neutrophil polarization and chemotaxis. Wt AQP9 and S11D AQP9 displayed a very dynamic distribution at the cell membrane, whereas the phosphorylation-deficient S11A AQP9 failed to localize to the plasma membrane. Furthermore, we found that Rac1 regulated the translocation of AQP9 to the plasma membrane. Our results show that AQP9 plays an active role in neutrophil volume regulation and migration. The display of AQP9 at the plasma membrane depends on AQP9 phosphorylation, which appeared to be regulated through a Rac1-dependent pathway.
Epithelial-specific knockout of the Rac1 gene leads to enamel defects
(Wiley, 2011-12) Huang, Z.; Kim, J.; Lacruz, R. S.; Bringas, P. Jr.; Glogauer, Michael; Bromage, T. G.; Kaartinen, V. M.; Snead, M. L.
The Ras-related C3 botulinum toxin substrate 1 (Rac1) gene encodes a 21-kDa GTP-binding protein belonging to the RAS superfamily. RAS members play important roles in controlling focal adhesion complex formation and cytoskeleton contraction, activities with consequences for cell growth, adhesion, migration, and differentiation. To examine the role(s) played by RAC1 protein in cell-matrix interactions and enamel matrix biomineralization, we used the Cre/loxP binary recombination system to characterize the expression of enamel matrix proteins and enamel formation in Rac1 knockout mice (Rac1(-/-)). Mating between mice bearing the floxed Rac1 allele and mice bearing a cytokeratin 14-Cre transgene generated mice in which Rac1 was absent from epithelial organs. Enamel of the Rac1 conditional knockout mouse was characterized by light microscopy, backscattered electron imaging in the scanning electron microscope, microcomputed tomography, and histochemistry. Enamel matrix protein expression was analyzed by western blotting. Major findings showed that the Tomes' processes of Rac1(-/-) ameloblasts lose contact with the forming enamel matrix in unerupted teeth, the amounts of amelogenin and ameloblastin are reduced in Rac1(-/-) ameloblasts, and after eruption, the enamel from Rac1(-/-) mice displays severe structural defects with a complete loss of enamel. These results support an essential role for RAC1 in the dental epithelium involving cell-matrix interactions and matrix biomineralization.
Nonactivated versus Thrombin-Activated Platelets on Wound Healing and Fibroblast-to-Myofibroblast Differentiation In Vivo and In Vitro
(Lippincott, Williams & Wilkins, 2012-01) Scherer, S. S.; Tobalem, M.; Vigato, E.; Heit, Y.; Modarressi, A.; Hinz, Boris; Pittet, B.; Pietramaggiori, G.
BACKGROUND: Platelet preparations for tissue healing are usually preactivated before application to deliver concentrated growth factors. In this study, the authors investigated the differences between nonactivated and thrombin-activated platelets in wound healing. METHODS: The healing effects (i.e., wound closure, myofibroblast formation, and angiogenesis) of nonactivated and thrombin-activated platelets were compared in experimental wounds in diabetic (db/db) animals. In vitro, fibroblast phenotype and function were tested in response to platelets and activated platelets. No treatment served as a negative control. RESULTS: Wounds treated with platelets reached 90 percent closure after 15 days, faster than activated platelets (26 days), and with higher levels of myofibroblasts and angiogenesis. In vitro, platelets enhanced cell migration and induced two-fold higher myofibroblast differentiation and contraction compared with activated platelets. CONCLUSIONS: Platelets stimulate wound healing more efficiently compared with activated platelets by enhancing fibroblast differentiation and contractile function. Similar levels of growth factors may induce different biological effects when delivered "on demand" rather than in an initial bolus.

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