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  • An In vitro investigation of vascular responses to dental resin component

    Maddux, William F.; Department of Oral Biology (Augusta University, 2002-04)
    Resin-based materials have become increasingly popular for use in dentistry. Components of these materials are known to leach out from placement sites, initially at high concentrations and thenafter at relatively lower levels. Available evidence indicates that dental resin components have detrimental effects towards gingival fibroblast and epithelial cells. However, despite the rich vascularity of the oral environment, there is only limited information on the effects of these substances on the responsiveness of vascular tissues. The present investigation was undertaken to determine the effects of the resin components, methyl methacrylate (MMA), hydroxyethyl methacrylate (HEMA), triethylene glycol dimethacrylate (TEGDMA) and dimethylaminoethyl methacrylate (DMAEMA), on vascular reactivity using the isolated rat aorta as a tissue model. In addition, possible vascular effects of the degradation products ofHEMA and DMAEMA, i.e., dimethylethanolamine (DME), methacrylic acid (MAA) and ethylene glycol (EG), were also assessed. In the investigation, isometric tension of aortic rings was measured, and various in vitro conditions were employed to study the mechanisms of resin-induced vascular reactivity. Biochemical assays of tissue levels of prostacyclin (PGh) and thromboxane Az (TXAz) were also carried out to determine if there was a correlation with the mechanical responses. It was found that MMA, HEMA, TEGDMA and DMAEMA caused concentration-dependant relaxation of NE-contracted rat aortic rings with and without endothelium. This finding suggests the existence of both endothelium-dependent and independent components for the vascular responses to these resin components. The endothelium-dependent but not independent responses were inhibited by N-nitro-Larginine methyl ester (L-NAME), indicating the involvement of endothelium-derived nitric oxide. The vasorelaxant effects of MMA, TEGDMA and DMAEMA on the endothelium-intact and denuded aortic rings were attenuated by indomethacin, providing evidence for the role of prostanoids, such as prostacyclin, in these responses. This observation was further supported by the increased production of prostacylin in response to TEGDMA, although this was not well established for MMA and DMAEMA. Glybenclamide selectively inhibited TEGDMA and DMAEMA-induced relaxation of the blood vessels with and without endothelium. However, while the effects of the inhibitor on TEGDMA-induced relaxation were similar in the endothelium intact and denuded aortae, they were greater in the intact tissues relaxed by DMAEMA. These results suggest the activation of vascular smooth muscle KATP channels by both TEGDMA and DMAEMA, and the possible release of endothelium-derived hyperpolarizing factor (EDHF) by DMAEMA. On the other hand, DMAEMA at a relatively high concentration caused contraction of the rat aorta while DME induced concentration-dependant contractile responsiveness at lower concentrations, in both the presence and absence of the endothelium. Endothelium removal did not alter the responses of the tissue to DME. The contractile effe?ts of DME were attenuated by indomethacin, indicating the involvement ofprotanoid metabolite(s) in the vascular action of this degradation product of DMAEMA. In support of this finding, there was enhanced production of TXA2 in aortic rings incubated with DME. Unlike DME, MAA, and EG were without effect on the rat aorta. It is concluded the MMA, HEMA, TEGDMA, and DMAEMA alter the function of blood vessels by causmg relaxation v1a different mechanisms, which, depending upon the substances under consideration, may at least involve the release of nitric oxide and prostacylin, and the activation ofKATP channels. Further, DMAEMA (at high concentration) and its degradation product DME, upon interaction with aortic tissue can cause vasoconstriction; the effect of DME may involve the generation of TXAz. These mechanisms may play a role in tissue homeostasis and certain pathophysiological conditions associated with the use of resin materials in dentistry.
  • Modulus of elasticity of demineralized dentin matrix

    Maciel, Keli Tenfuss; Department of Oral Biology & Physiology (Augusta University, 1995-05)
  • Immunohistochemical evaluation of labial salivary glands in xerostomic patient

    Ma, Yat-Ho; Medical College of Georgia (Augusta University, 2011-12)
  • The effects of fibronectin and the rgd binding sequence on gingival fibroblasts in an in vitro wound healing mode

    Lyons, James Cornelius; Medical College of Georgia (Augusta University, 2004-04)
    The purpose of this study was to ip.vestigate the influence of fibronectin or the tripeptide Arginine-Glycine-Aspartate (RGD) on human gingiyal fibroblast ceil migration and wound repopulation using an in vitro wound-healing model. The hypothesis was that increasing concentrations of .either fibroneetin or RGD would positively enhance the .. ability of human fibroblast ceiis to repopulate a wound over time. Fibronectin in concentrations of 5 ~tg, 10 )lg, arid- 15 Jlg/ ml and RGD concentrations of 1 TJg, 4 11g, 8 TJg, and 12 11g/ml were used in this study. The in vitro wound-healing model utilized 12-well culture plates in which gingival fibroblasts were seeded and grown to confluence. A 24-hour synchronization phase preceded the creation of a linear 3 mm wide cell-free zone across the diameter of each well. The cell-containing wells were replenished daily with a serum-free medium containing either fibronectin or RGD at the specified concentrations. Control samples received serum-free media only. Cell samples were then fixed and stained with either crystal violet or hematoxylin & eosin every 48-hours for eight days. Photomicrographs were taken of each stained well for the histomorphometric examination and measurements of wound fill using the Scion Image Analysis Software. In follow-up experiments, using the same procedures, the 3 mm wide cell-free zone was coated with a RGD-polyol substrate immediately after wounding. The polyols were mixed with RGD to concentrations of either 1 T]g, 4 TJg, 8 T]g, or 12 T]g/ml. Positive controls were coated with an RGD-free polyol substrate while the negative controls were coated with serum-free media only. All cells received daily nutrient replenishment and were subsequently fixed, stained, photographed, examined and measured as previously mentioned. Conclusions reached in this study were as follows: Both the fibronectin and RGD challenge appeared to result in positive dose-dependent responses in the early days of wound repopulation when compared to controls. 1. The serum-free defined media provides an excellent nutrition source for gingival fibroblast cells. 2. All experimental concentrations of fibronectin appeared to stimulate the migration of gingival fibroblasts into the wound area up to day two. Fibroblast cells exposed to fibronectin at 15ug/ml consistently displayed more wound fill. 3. All experimental concentrations of RGD also appeared to positively influence fibroblast migration through day two. 4. All RGD-polyol samples appeared to support better wound fill than the positive control (polyol only) but did not appear to improve fibroblast migration when compared to the negative control (serum-free media only).
  • The effect of dental-curing light on in vitro epithelial cell growth

    Lyons, Collns Thomas; Department of Oral Biology (Augusta University, 2002-01)
  • The Effects of Human Gingival Epithelial Cells on Tenascin Production by Human Gingival Fibroblasts

    Linatoc, A John; Department of Oral Biology (Medical College of Georgia, 1993-02)
    The goals of periodontal therapy are to encourage healing through regeneration of lost periodontal tissue, restore biologic function, improve esthetics, and provide comfort. Fibroblasts are the principal cell type in the connective tissues of the periodontium and they perform important functions in development, physiology, and healing. Extracellular matrix proteins, such as tenascin, are secreted by fibroblasts and may play an important role in morphogenesis and cel,l differentiation. A better understanding of cellular interactions may facilitate the development of predictable regenerative periodontal procedures. The purpose of this study was to examine tenascin localization in human gingival tissues and cultures of two types of human periodontal fibroblasts. Co-cultures of periodontal fibroblasts and human gingival epithelial cells were also produced and examined for tenascin localization. In addition, the effects of medium and time on tenascin secretion by cultured periodontal fibroblasts were quantitatively analyzed by computer assisted densitometric analysis. The results indicate that tenascin is localized at the interface between the overlying epithelium and the connective tissue papilla. Co-cultures failed to show the same tenascin staining pattern as was found in the gingival tissues. No significant difference in tenascin secretion was found between cultured gingival or periodontal ligament fibroblasts. Medium containing 10% fetal bovine serum (FBS) stimulated a significant increase in tenascin production from 48hrs to 96hrs (p50.0405) as compared to serum free medium (p50.0001) or medium conditioned by human gingiv~l fibroblasts (p50.0031), hamster cheek pouch epithelial cells (p50.0001), and human gingival epithelial cells (ps0.02880). It appears that the cell culturing techniques, tissue explant characteristics, the quality and quantity of soluble serum, and/or inducing factors may have influenced the observed pattern of tenascin secretion.
  • Enos Regulation by Phosphorylation and Protein-Protein Interactions

    Li, Chunying; Vascular Biology Center (Medical College of Georgia, 2006-08)
    Endothelial nitric oxide synthase ( eNOS) catalyzes the conversion of L-arginine to Lcitrulline and nitric oxide (NO). Protein phosphorylation and protein-protein interactions are two major mechanisms for eNOS regulation at the post-translational level, three aspects of which have been investigated in this study. The first aspect of eNOS regulation that we have examined is whether endos\atin (ES) is a novel eNOS-activating agonist responsible for stimulating multi-site eNOS phosphorylation in endothelial cells. We show that ES induces acute endothelial NO release accompanied by eNOS phosphorylation events in cultured bovine aortic endothelial cells (BAECs ). ES also induces relaxation of rat aortic rings. The second aspect of eNOS regulation that we have examined is the role of individual eNOS serine and threonine phosphorylation sites in the regulation of eNOS activity in BAECs. We mutated all five Thr- and Ser- sites of eNOS phosphorylation to aspartate or alanine and overexpressed the proteins in BAECs using adenoviral-mediated gene transfer. We show that mimicking phosphorylation of Ser-116 and Thr-497 is inhibitory, and mimicking phosphorylation of Ser-617, Ser-635 and Ser- 1179 is stimulatory. Mimicking phosphorylation of Ser-635 and Ser-1179 together does not show synergistic effects on endothelial NO release. In addition, removal of any of the five Ser/Thr phosphorylation sites does not affect thapsigargin- or VEGF-stimulated NO release. A final aspect of eNOS regulation that we have investigated is the role of proteinprotein interactions of eNOS with the CAT (cationic amino acid transporter)-! arginine transporter. We show that eNOS interacts directly with CAT-1 and that overexpression of CAT-I proteins in BAECs results in significant increases in NO release which is not altered by the CAT-I inhibitor, L-lysine, suggesting that NO production in this in vitro model is independent of CAT-I mediated arginine transport. Furthermore, eNOS enzymatic activity is increased in lysates ofCAT-I-overexpressing cells accompanied by increased eNOS association with CAT-I, alterations of eNOS phosphorylation and eNOS association with caveolin-1. The present study adds to the knowledge of the regulation of eNOS by multi-site phosphorylation and protein-protein interactions.
  • Therapeutic Targeting of P2X7 after Traumatic Brain Injury

    Kimbler Jr, Donald E; Department of Neuroscience and Regenerative Medicine (Georgia Health Sciences University, 2012-02)
    Traumatic brain injury (TBI) is a leading cause of death and disability worldwide. Cerebral edema, the abnormal accumulation of fluid within the brain parenchyma, contributes to elevated intracranial pressure (ICP) and is a common life-threatening neurological complication following TBI. Unfortunately, neurosurgical approaches to alleviate increased ICP remain controversial and medical therapies are lacking due in part, to the absence of viable drug targets. In the present study, genetic inhibition (P2X7-/- mice) of the purinergic P2x7 receptor attenuated the expression of the pro-inflammatory cytokine, interleukin-I~ (IL-1~) and reduced cerebral edema following controlled cortical impact, as compared to wild-type mice. Similarly, the clinically useful P2X7 inhibitor, brilliant blue G (BBG), inhibited the expression of IL-1~, limited edemic development and prevented the development of post-traumatic depression and anxiety. The beneficial effects of BBG were observed following either prophylactic administration via the drinking water for one week prior to injury or via an intravenous bolus administration up to four hours after TBI, suggesting a clinically-implementable therapeutic window. Notably, P2X7 localized within astrocytic end feet and administration of BBG decreased the expression of glial fibrillary acidic protein (GFAP), a reactive astrocyte marker, and reduced the expression of aquaporin-4 (AQP4), an astrocytic water channel that promotes cellular edema. Together, these data implicate P2X7 as a novel therapeutic target to prevent secondary neurological injury after TBI, a finding that warrants further investigation.
  • The Effects of Myostatin Deficiency on Fracture Healing in a Rodent Model

    Kellum, Ethan Lasha; Department of Cellular Biology & Anatomy (Medical College of Georgia, 2008-04)
    Myostatin (GDF-8) is a negative regulator of skeletal muscle growth; mice lacking myostatin show increased muscle mass and enhanced muscle regeneration. It has previously been shown that myostatin deficiency increases bone strength and biomineralization throughout the skeleton. This study tests the hypothesis that myostatindeficiency enhances fracture healing using a fibula osteotomy model. Adult wild-type mice, mice heterozygous for the myostatin mutation, and mice homozygous for the disrupted myostatin sequence were included for study at two- and four-weeks following the osteotomy procedure. Fracture healing was assessed using radiographic, histological, and biomechanical techniques. The fracture callus of myostatin-deficient animals is significantly larger than that of wild-type mice at two- and four-weeks post-osteotomy, and also shows increased total bone area within the callus at each time point. Mechanical testing demonstrates that the fracture callus of myostatin-deficient mice exhibits significantly greater peak force and energy to fracture (toughness) compared to the callus of normal mice. Myostatindeficient animals also have a significantly greater number of regenerative myofibers surrounding the fracture callus compared to normal mice. Results presented here show that the improved muscle regeneration and osteogenic differentiation that accompany myostatin deficiency also enhance bone repair following injury. These findings suggest that myostatin inhibitors may serve as potential therapeutic agents for accelerating muscle and bone healing in orthopaedic trauma cases where significant damage to both muscle and bone has occurred.
  • Reducing Noise in Neonatal Intensive Care Nurseries: Incubator Covers

    Kellam, Barbara Cruit; College of Nursing (Medical College of Georgia, 2005-04)
    Minimal safe noise levels for premature infants are not definitively known. The American Academy of Pediatrics (1997) set a standard of 50 decibels maximum for hourly noise levels in NICUs to protect the normal growth and development of pre-term infants. Disruption of normal sensorineural development in preterm infants may result from exposure to environmental noise during hospitalization. Concerns emerge from a literature review of noise measurement studies in Neonatal Intensive Care Units. First, few studies have measured high frequency sound within occupied incubators. Second, interventions to reduce noise exposure to preterm infants cared for in incubators focused on transient physiological and/or behavioral states. Third, intervention studies designed to reduce noise within incubators indicated conflicting results. Finally, none of the published studies used acoustical products as the core of an incubator cover. This experiment measured noise levels within the preterm infant's incubator under the treatment and the control condition. It was one of the few studies to perform sound spectral analysis while subjects were cared for within incubators. It was the first study to test the effectiveness of an incubator cover with a Noise Reduction Coefficient of 0;95: Acoustical Incubator Covers©. This study indicated that Acoustical Incubator Covers© significantly reduced high frequency sound(~ 1000 Hz). When examined using ANCOVA procedures, the data indicated a trend toward improved weight gain in the group that received Acoustical Incubator Covers©. There was no difference between the two groups on the variable of pass rate on the hearing screening exam. Length of hospitalization differed between the two groups by 10 days with the treatment group having the lower mean length of hospitalization. Further study with a larger sample size is needed to determine if reduced high frequency noise promotes better weight gain and fewer hospital days in preterm infants.
  • Role of carboxypeptidase E and bradykinin in the regulation of GnRH and LH secretion

    Liu, Tongyun; Medical College of Georgia (Augusta University, 2000-07)
  • Notch3 signaling mediates heterotypic cell interatcions during blood vessel formation

    Liu, Hua; Medical College of Georgia (Augusta University, 2010-11)
  • Astrocytes Modulate Endothelial Cell Growth, Differentiation, and Fibronectin Expression

    Jiang, Baoen; Department of Cellular Biology & Anatomy (Medical College of Georgia, 1993-08)
    The retinal vascular development in the nerve fiber layer occurs by in situ differentiation of vascular precursor cells, but the mechanisms controlling this process are still unknown. We sought to determine the stimuli regulating. the organization of vascular precursor cells into retinal vessels by analyzing the roles of extracellular matrix and astrocytes. Immunolocalization analyses in the developing rat show that rat retinal vessels form as a polygonal network, beginning at the optic disc and extending to the ora. serrata by pll. Factor VIII-positive endothelial cells and laminin- and fibronectin-positive basement membrane codistribute with lumenized vessels. GFAP-positive astrocytic processes associate closely with the formed vessels, but ~re also present between the vessels. In the avascular area just distal to the forming vessels, GFAP-positive astrocytes form a fibronectin-positive polygonal meshwork pattern that resembles the immature vascular network. Laminin is not expressed in this area. In the area of the astrocyte-fibronectin meshwork, Bandierea Griffonia Simplifolia I B4 lectin labeled vascular precursor cells elongate and align along the astrocyte processes. Double label analysis indicates that both astrocytes and vascular . cells produce fibronectin. In situ hybridization shows that fibronectin mRNA expression is heaviest in the meshwork areas, suggesting that fibronectin synthesis is required in the early stage of retinal vasculogenesis. 0~11 culture experiments confirmed a strong influence of astrocytes on retinal microvascular endothelial cell growth, morphological differentiation, and extracellular matrix expression. Immunolocalization studies of cultured cells suggested and Western blot and Northern blot analyses confirmed a significant increase in fibronectin expression, when endothelial cells were grown in conditioned medium (CM) from astrocytes as compared with controls grown in CM from endothelial cells or pericytes. When retinal microvascular endothelial cells were grown in CM from astrocyte I endothelial cell co-culture, their growth was significantly inhibited as compared with controls grown in normal growth medium or CM from endothelial cells, astrocytes, or a mixture of 50% astrocyte CM/50% endothelial cell CM. In addition, endothelial cells grown in CM from astrocyte I endothelial cell co-culture elongated and became aligned in capillary-like structures. Endothelial cells grown in CM from astrocytes, endothelial cells, or a mixture of 50%astrocyte CM/50% endothelial cell CM alone retained normal· cobblestone morphology. CM from astrocytes alone stimulates retinal microvascular endothelial cell growth as compared with CM from endothelial cells, astrocyte I endothelial cell co-culture, or a mixture of 50% astrocyte CM/50% endothelial cell CM. These data indicate that rat retinal vascular development occurs by in situ differentiation of vascular precursor cells in close contact with astrocytes. Moreover, astrocytes modulate retinal microvascular endothelial cell growth, morphological differentiation, and fibronectin expression by release of growth factor-like substances. At least two factors are involved - one that requires astrocyte/ endothelial cell contact for activity and one that does not.
  • Generation of a Retroviral Vector Expressing an Anti-HIV-1 TAT Hammerhead Ribozyme as Part of a LACZ mRNA and Its Effect on HIV Replication

    Jackson, William H.; Department of Cell and Molecular Biology (Medical College of Georgia, 1995-08)

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