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#21 Title: Proteinase Activities in Co-infections of Cariogenic Bacteria D PHAN (1), X WEI (1), L ZHU (1), C LE (2), G MARSHALL (2), S MARSHALL (2), J D B FEATHERSTONE (2), L ZHAN (1) (2), W LI (1) (1) UCSF Department of Orofacial Sciences and (2) UCSF Department of Preventive and Restorative Dental Sciences Objective: Dentin caries formation involves loss of both demineralization and degradation of organic matrix, mainly consisting of collagen. Mutans Streptococci (MS) and Lactobacillus (LB) are two major species of caries inducing bacteria. MS and LB were frequently isolated in co-existence from dental plaque on caries-active sites in dentin. Previous studies have found collagenase-like activity in MS produced but were inconclusive in LB. Similar to MS, our recent study identified collagenase-like activities in LB. This study aimed to identify the effect of interactions of secretive proteins from MS and LB on their collagenase-like activity. Methods: MS and LB were individually cultivated in BHI broth at 37°C under anaerobic condition for 48 hours. The collagenase-like activity in the individual culture supernatant of MS, LB or mixture of the two at 1:1 ratio, adjusted for cell density of the culture at OD600, was evaluated and compared by zymography and DQ-collagen assays. Results: The zymography showed that both LB and MS produced collagenase-like proteases with similar enzymatic profiles. The collagenase-like activities were further confirmed by using DQ-collagen as a substrate. LB exhibited the stronger activity than MS. When the two culture supernatants were mixed at 1:1 ratio, the collagenase-like activity was significantly reduced compared to the individual supernatants alone. Conclusion: The decreased proteinase activity in the mixture of culture supernatants of LB and MS suggests that the secretions from MS and LB may produce substance that inhibit the collagenase-like activity of the other bacteria or their secretive proteins may interact to form inhibitory molecules, resulting in less proteinase activities. Further study will be needed to identify the nature of the collagenase-like protease and the interaction of MS and LB on their collagenase-like activity. This property may play an important role on the balance of caries progression in dentin. Support: NIDCR Grants R01DE015821 and U54DE019285 #22 Title: Accelerated implant osseointegration via liposomal Wnt3a S ROOKER (1), A POPELUT (2), P LEUCHT (2), M MEDIO (2), J BRUNSKI (2), J HELMS (2) (1) UCSF School of Dentistry (2) Department of Surgery, Stanford School of Medicine Introduction: Dental implants are prosthetic replacements for teeth that have been damaged or lost due to disease. The most successful types of dental implants are those in which bone matrix is in direct contact with the surface of the implant, a condition known as osseointegration. In an effort to promote osseointegration, implant surfaces have undergone major alterations but the majority of these modifications have only resulted in modest improvements. The use of protein-based approaches to stimulate implant osseointegration has also been under intense investigation: Bone morphogenetic proteins induce endochondral ossification in skeletal fractures but their use is contraindicated for implant osseointegration. Objective: We aimed to utilize an alternative protein-based strategy to stimulate implant osseointegration, based on the welldocumented role for Wnt signaling in bone regeneration. Methods: A needle was placed as an implant into the tibiae of Wnt reporter mice, and using a formulation of the Wnt3a protein that retains its biological activity in vivo, we injected L-Wnt3a around the implant site three days after placement. Bone formation around the implant was examined by multiple time points, using histologic staining and histomorphometric analyses; immunohistochemistry, and in situ hybridization for osteogenic markers. Results: We found that a single injection resulted in exuberant proliferation of osteoprogenitor cells in the bone marrow cavity, followed by the accelerated differentiation into matrix-secreting osteoblasts. L-Wnt3a induced 350% more peri-implant bone formation than controls. Conclusion: we demonstrated that delivery of L-Wnt3a augments bone formation around implants. We envision this strategy can be employed to accelerate implant osseointegration and ultimately improve the functional lifespan of dental implants. Support: This work was funded by CIRM TR1-01249. Support: CIRM TR1-01249 #23 Title: Therapeutic use of Trophoblastic Stem Cells to promote bone regeneration in fracture repair A SADAT (1) (2), C BAHNEY (2), E MALTEPE (3) (4), R MARCUCIO (2) (1) UCSF School of Dentistry (2) Orthopaedic Trauma Institute, San Francisco General Hospital (3) UCSF Department of Pediatrics (4) UCSF Department of Neonatology Introduction: Insufficient blood supply is the leading cause of impaired fracture healing. The rate of delayed or non-unions is 46% in patients with concomitant vascular injuries, compared to only 10% in the general population. This suggests that enhancing the vasculature at the fracture site may improve the rate or extent of fracture repair. During embryonic development, vascular remodeling of the maternal blood supply is mediated by endovascular invasion of Trophoblastic Stem Cells (TSCs) to create a high volume, low pressure flow rate designed to increase blood to the developing embryo. Objectives: This study investigates a novel therapeutic strategy to temporarily increase the local blood supply at the fracture site by delivering TSCs immediately following fracture. We hypothesize that TSCs will promote vascular remodeling and enhance fracture repair. School of Dentistry Research and Clinical Excellence Day 2011 Program 11
Methods: Non-stable fractures were created mid-diaphysis in the murine tibia and administered an injection of murine-derived TSCs expressing enhanced Green Fluorescent Protein (eGFP) or a control media. Bone healing and vascularity were analyzed histologically on days 5, 7, 14, 21. Results: Preliminary data suggests that TSCs may promote vascular remodeling during the early phases of fracture repair as evident from blood islands near the fracture site at day 5 that resemble those seen in the placenta. Furthermore, TSC injected mice reveal a fracture callus predominately composed of bone at day 14, compared to controls containing both cartilage and bone. Conclusions: This evidence suggests that a local injection of TSCs at a non-stable fracture may remodel the local vasculature and enhance the rate of fracture repair by increasing blood supply. Alternatively the TSCs may have accelerated bone regeneration by providing trophic cytokines at the fracture. Further experiments are aimed at determining the specific mechanism by which TSCs contribute to vascular remodel ing and enhanced osteo-regeneration. Support: This project was supported by NIH/NCRR/OD UCSF-CTSI Grant Number TL1 RR024129. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH. #24 Title: Role of Nance-Horan Syndrome protein in epithelial morphogenesis R SARMICANIC (1), T WITTMANN (2), P KUMAR (2) (1) UCSF School of Dentistry, (2) UCSF Department of Cell and Tissue Biology Introduction: Null mutations in the NHS gene cause an X-linked genetic disorder, Nance-Horan Syndrome that is characterized by congenital cataract, dental abnormalities, and in few cases, mental retardation. Many of these phenotypes could be explained by defects in epithelial morphogenesis. NHS has been proposed to interact with the tight junction protein ZO-1 and regulate actin dynamics through an N-terminal WAVE domain. However, the function of NHS during epithelial morphogenesis or remodeling is unknown. Objective: To begin to understand NHS function in epithelial cells, the current study examined the localization and dynamics of full length NHS protein in a 3D epithelial cell culture model. Methods: An MDCK cell line stably expressing EGFP-NHS was generated, and EGFP-NHS dynamics were investigated by live cell spinning disk confocal microscopy in 3D cell culture in which MDCK cells form apical-basal polarized, spherical cysts. Fluorescence Recovery Afte r Photobleaching (FRAP) was used to determine the NHS turnover at cell-cell junctions. Results: EGFP-NHS localized to lateral cell-cell contacts in polarized epithelial cells, but was absent from the basal or apical cell surfaces. EGFP-NHS localization was distinctly different from ZO-1. This was different from previously reported colocalization of ZO-1 and NHS, and indicates that NHS is not involved in tight junction formation. However, EGFP-NHS partially co-localized with actin cables that run laterally between polarized epithelial cells. NHS turnover at cell-cell junctions was measured to have an average t1/2 of 6.7±1.8s. This time was faster than t1/2 of 119±21s; the recovery time previously reported for ZO-1. The recovery time for NHS further indicated that NHS is not a tight junction protein. Conclusion: Our results support the hypothesis that NHS is an integral part of cell-cell contacts in polarized epithelial cells, where it may regulate circumferentia l actin polymerization dynamics. Support: American Associate of Dental Research (AADR) Student Research Fellowship and support from Delta Dental of California #25 Title: The Role of NMHCIIA in Fusion of the Secondary Palate V SINGH (1) J BUSH (2) (1) UCSF School of Dentistry, (2) UCSF Department of Cell and Tissue Biology and the Program in Craniofacial and Mesenchymal Biology Introduction: Non-syndromic cleft palate is a common craniofacial anomaly, occurring in about 1/1000 births worldwide. Disruption in elevation, attachment, or fusion of secondary palatal shelves during development can cause clefts, but the molecular effectors of this morphogenesis remain unclear. Gene association studies have identified MYH9, encoding the heavy chain of non-muscle myosin-IIA (NMHCIIA), as a candidate in non-syndromic clefting. Since non-muscle myosin-II (NMII) functions in numerous cell biological processes including cell migration, adhesion, and cytokinesis, we sought to pinpoint the role of NMHCIIA in palatogenesis. Objectives: To examine the role of NMII in palatal shelf fusion by expression analysis and functional approaches. Methods: Expression analysis was performed on mouse embryo sections at embryonic day 13.5 (E13.5) and 14.5 (E14.5) using insitu hybridization (ISH) and immuno-fluorescence following standard protocols. For functional analysis, palatal shelves were dissected at E13.5 and placed in BGjb medium containing DMSO control or 10μM blebbistatin (an inhibitor of NMII) in DMSO at 37°C/5% CO2 (96hrs). After coronal sectioning (7μm) and hematoxylin/eosin staining, palatal fusion was quantified by mean fusion score (MFS). Results: ISH and immuno-fluorescence analysis revealed elevated expression of NMHCIIA in medial edge epithelium during fusion. MFS suggested partial fusion(5/7) and nonfusion(2/7) for palatal shelves cultured in blebbistatin medium(n1=7). Shelves in DMSO medium(n2=8) showed partial fusion(3/8) and complete fusion(5/8) using MFS. Differences in fusion between two conditions were significant by Mann-Whitney U-test (p
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