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Purpose: The aim of this study was to evaluate the surface characteristics and gingival fibroblast adhesion of disks composed of implant and abutment materials following brief and repeated instrumentation with instruments commonly used in procedures for implant maintenance, stage-two implant surgery, and periimplantitis treatment. Materials and Methods: One hundred twenty disks (40 titanium, 40 titaniumzirconium, 40 zirconia) were grouped into treatment categories of instrumentation by plastic curette, titanium curette, diode microlaser, rotary titanium brush, and no treatment. Twenty strokes were applied to half of the disks in the plastic and titanium curette treatment categories, while half of the disks received 100 strokes each to simulate implant maintenance occurring on a repetitive basis. Following analysis of the disks by optical laser profilometry, disks were cultured with human gingival fibroblasts. Cell counts were conducted from scanning electron microscopy (SEM) images. Results: Differences in surface roughness across all instruments tested for zirconia disks were negligible, while both titanium disks and titaniumzirconium disks showed large differences in surface roughness across the spectrum of instruments tested. The rotary titanium brush and the titanium curette yielded the greatest overall mean surface roughness, while the plastic curette yielded the lowest mean surface roughness. The greatest mean cell counts for each disk type were as follows: titanium disks with plastic curettes, titanium-zirconium disks with titanium curettes, and zirconia disks with the diode microlaser. Conclusion: Repeated instrumentation did not result in cumulative changes in surface roughness of implant materials made of titanium, titanium-zirconium, or zirconia. Instrumentation with plastic implant curettes on titanium and zirconia surfaces appeared to be more favorable than titanium implant curettes in terms of gingival fibroblast attachment on these surfaces.

Lysophosphatidic acid (LPA) is a phospholipid mediator that plays multiple cellular functions by acting through G protein-coupled LPA receptors. LPAs are known to be key mediators in inflammation, and several lines of evidence suggest a role for LPAs in inflammatory periodontal diseases. A simple and sensitive liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method has been developed and validated to quantify LPA species (LPA 18:0, LPA 16:0, LPA 18:1 and LPA 20:4) in human saliva and gingival crevicular fluid (GCF). LPA 17:0 was used as an internal standard and the LPA species were extracted from saliva by liquid-liquid extraction using butanol. Chromatography was performed using a Macherey-Nagel NUCLEODUR® C8 Gravity Column (125 mm × 2.0 mm ID) with a mixture of methanol/water: 75/25 (v/v) containing 0.5% formic acid and 5 mM ammonium formate (mobile phase A) and methanol/water: 99/0.5 (v/v) containing 0.5% formic acid and 5mM ammonium formate (mobile phase B) at a flow rate of 0.5 mL/min. LPAs were detected by a linear ion trap-triple quadrupole mass spectrometer with a total run time of 8.5 min. The limit of quantification (LOQ) in saliva was 1 ng/mL for all LPA species and the method was validated over the range of 1-200 ng/mL. The method was validated in GCF over the ranges of 10-500 ng/mL for LPA 18:0 and LPA 16:0, and 5-500 ng/mL for LPA 18:1 and LPA 20:4. This sensitive LC-MS/MS assay was successfully applied to obtain quantitative data of individual LPA levels from control subjects and patients with various periodontal diseases. All four LPA species were consistently elevated in samples obtained from periodontal diseases, which supports a role of LPAs in the pathogenesis of periodontal diseases.

Migration of fibroblasts plays an essential role in tissue repair after injury. Sphingosine 1-phosphate (S1P) is a multifunctional mediator released by many cells that can be released in inflammation and after injury. This study evaluated the effect of S1P on fibroblast chemotaxis toward fibronectin. S1P alone did not affect fibroblast migration, but S1P enhanced fibronectin-directed chemotaxis in a concentration-dependent manner. The effect of S1P was not mimicked by dihydro (dh) S1P or the S1P(1) receptor agonist SEW2871. S1P augmentation of fibroblast chemotaxis, however, was completely blocked by JTE-013, an S1P(2) antagonist, but not by suramin, an S1P(3) antagonist. Suppression of the S1P(2) receptor by small interfering (si)RNA also completely blocked S1P augmentation of fibroblast chemotaxis to fibronectin. S1P stimulated Rho activation and focal adhesion kinase (FAK) phosphorylation, and these were also significantly inhibited by the S1P(2) receptor antagonist (JTE-013) or by S1P(2) siRNA. Further, the potentiation of S1P signaling was blocked by the Rho-kinase inhibitor Y-27632 in a concentration-dependent manner. Inhibition of FAK with siRNA reduced basal chemotaxis toward fibronectin slightly but significantly, and almost completely blocked S1P augmented chemotaxis. These results suggest that S1P-augmented fibroblast chemotaxis toward fibronectin depends on the S1P(2) receptor and requires Rho and Rho-kinase, and FAK phosphorylation. By augmenting fibroblast recruitment, S1P has the potential to modulate tissue repair after injury. The pathways by which S1P mediates this effect, therefore, represent a potential therapeutic target to affect tissue repair and remodeling.

Enhanced proliferation of airway smooth muscle is thought to contribute to the pathogenesis of asthma and other obstructive airway diseases. Lysophosphatidic acid (LPA) is a simple bioactive lipid mediator that stimulates mitogenesis in fibroblasts and some other cell types. The effects of LPA on mitogenesis of cultured human airway smooth muscle cells were determined by measuring [3H]thymidine incorporation into cellular DNA. LPA induced a concentration-dependent stimulation of [3H]thymidine incorporation of a similar magnitude to that induced by serum, with the effects of 50 microM LPA being similar to those of 5% serum. Stimulation by LPA and by serum was almost completely eliminated in cells exposed to pertussis toxin, indicating involvement of a pertussis toxin-sensitive G protein in mitogenic signaling by these agents. Epidermal growth factor (EGF) induced stimulation of a similar magnitude as that with LPA, but the stimulation by EGF was insensitive to pertussis toxin. LPA and EGF, when added together, exhibited a markedly synergistic stimulation of [3H]thymidine incorporation that was typically 10-fold greater than the stimulation with either agent alone. LPA and EGF also stimulated mitogenesis assessed by cell growth, and again LPA and EGF together exhibited synergism. These results suggest the possibility that stimulation of airway smooth muscle cell proliferation by LPA, either alone or by enhancing effects of other growth factors, could play a role in normal airway remodeling or in the pathological proliferation of smooth muscle in various airway diseases.

Many, but not all, zinc-containing neurons in the brain are a subclass of the glutamatergic neurons, and they are found predominantly in the telencephalon. These neurons store zinc in their presynaptic terminals and release it by a calcium-dependent mechanism. These "vesicular" pools of zinc are viewed as endogenous modulators of ligand- and voltage-gated ion channels. Metallothioneins (MTs) are low molecular weight zinc-binding proteins consisting of 25-30% cysteine, with no aromatic amino acids or disulfide bonds. The areas of the brain containing high contents of zinc such as the retina, the pineal gland, and the hippocampus synthesize unique isoforms of MT on a continuous basis. The four MT isoforms are thought to provide the neurons and glial elements with mechanisms to distribute, donate, and sequester zinc at presynaptic terminals; or buffer the excess zinc at synaptic junctions. In this cause, glutathione disulfide may participate in releasing zinc from MT. A similar nucleotide and amino acid sequence has made it difficult to obtain cDNA probes and antibodies capable of distinguishing indisputably among MT isoforms. MT-I and MT-II isoforms are found in the brain and in the peripheral tissues; MT-III isoform, possessing an additional seven amino acids, is expressed mostly in the brain and to a very minute extent in the intestine and pancreas; whereas MT-IV isoform is found in tissues containing stratified squamous epithelial cells. Since MTs are expressed in neurons that sequester zinc in their synaptic vesicles, the regulation of the expression of MT isoforms is extremely important in terms of maintaining the steady-state level of zinc and controlling redox potentials. The concentration of zinc has been shown to be altered in an extensive number of disorders of the central nervous system, including alcoholism. Alzheimer-type dementia, amyotrophic lateral sclerosis, Down's syndrome, epilepsy, Friedreich's ataxia, Guillaine-Barre syndrome, hepatic encephalopathy, multiple sclerosis, Parkinson's disease, Pick's disease, retinitis pigmentosa, retinal dystrophy, schizophrenia, and Wernicke-Korsakoff syndrome. The status of MT isoforms and other low molecular weight zinc-binding proteins in these conditions, diseases, disorders, or syndromes is being delineated at this time. Since several of these disorders, such as amyotrophic lateral sclerosis, are associated with oxidative stress, and since MT is able to prevent the formation of free radicals, it is believed that cytokine-induced induction of MT provides a long-lasting protection to avert oxidative damage.