Effect of niobium content on the microstructure and thermal properties of fluorapatite glass-ceramics.

Niobium oxide has been shown to improve biocompatibility and promote bioactivity. The purpose of this study was to evaluate the effect of niobium oxide additions on the microstructure and thermal properties of fluorapatite glass-ceramics for biomedical applications. Four glass-ceramic compositions with increasing amounts of niobium oxide from 0 to 5 wt % were prepared. The glass compositions were melted at 1,525 degrees C for 3 h, quenched, ground, melted again at 1,525 degrees C for 3 h and furnace cooled. The coefficient of thermal expansion was measured by dilatometry. The crystallization behavior was evaluated by differential thermal analysis. The nature of the crystalline phases was investigated by X-ray diffraction. The microstructure was studied by SEM. In addition, the cytotoxicity of the ceramics was evaluated according to the ASTM standard F895--84. The results from X-ray diffraction analyses showed that fluorapatite was the major crystalline phase in all glass-ceramics. Differential thermal analyses revealed that fluorapatite crystallization occurred between 800 and 934 degrees C depending on the composition. The coefficient of thermal expansion varied from 7.6 to 9.4 x 10(-6)/ degrees C. The microstructure after heat treatment at 975 degrees C for 30 min consisted of submicroscopic fluorapatite crystals (200--300 nm) for all niobium-containing glass-ceramics, whereas the niobium-free glass-ceramic contained needle-shaped fluorapatite crystals, 2 microm in length. None of the glass-ceramics tested exhibited any cytotoxic activity as tested by ASTM standard F895--84.

Modulation of gingival fibroblast minocycline accumulation by biological mediators.

Gingival fibroblasts actively accumulate tetracyclines, thereby enhancing their redistribution from blood to gingiva. Since growth factors and pro-inflammatory cytokines regulate many fibroblast activities, they could potentially enhance fibroblast minocycline accumulation. To test this hypothesis, we treated gingival fibroblast monolayers for 1 or 6 hours with platelet-derived growth factor-BB (PDGF), fibroblast growth factor-2 (FGF), transforming growth factor-beta1 (TGF), or tumor necrosis factor-alpha (TNF). Minocycline uptake was assayed at 37 degrees by a fluorescence method. All 4 factors significantly enhanced minocycline uptake (P < or = 0.008, ANOVA), primarily by increasing the affinity of transport. Treatment for 6 hours with 10 ng/mL FGF, PDGF, TGF, or TNF enhanced fibroblast minocycline uptake by 19% to 25%. Phorbol myristate acetate enhanced fibroblast minocycline uptake by 28%, suggesting that protein kinase C plays a role in up-regulating transport. These effects on transport provide a mechanism by which systemic tetracyclines could be preferentially distributed to gingival wound or inflammatory sites.

Accumulation of ciprofloxacin and minocycline by cultured human gingival fibroblasts.

Through a mechanism that is unclear, systemic fluoroquinolones and tetracyclines can attain higher levels in gingival fluid than in blood. We hypothesized that gingival fibroblasts take up and accumulate these agents, thereby enhancing their redistribution to the gingiva. Using fluorescence to monitor transport activity, we characterized the accumulation of fluoroquinolones and tetracyclines in cultured human gingival fibroblast monolayers. Both were transported in a concentrative, temperature-dependent, and saturable manner. Fibroblasts transported ciprofloxacin and minocycline with K(m) values of 200 and 108 micro g/mL, respectively, at maximum velocities of 4.62 and 14.2 ng/min/ micro g cell protein, respectively. For both agents, transport was most efficient at pH 7.2 and less efficient at pH 6.2 and 8.2. At steady state, the cellular/extracellular concentration ratio was > 8 for ciprofloxacin and > 60 for minocycline. Thus, gingival fibroblasts possess active transporters that could potentially contribute to the relatively high levels these agents attain in gingival fluid.

Mechanisms of fluoroquinolone transport by human neutrophils.

Neutrophils accumulate ciprofloxacin and other fluoroquinolones, a process that enhances the killing of intracellular pathogens and could facilitate the delivery of these agents to infection sites by migrating neutrophils. The mechanisms by which transport occurs have not been characterized. In the present study, quiescent neutrophils transported ciprofloxacin with an observed K(m) of 167 microgram/ml (501 microM) and a maximum velocity of 25.2 ng/min/10(6) cells. When neutrophils were stimulated with phorbol myristate acetate (PMA), a second component of ciprofloxacin transport was induced. This pathway had an apparent K(m) of 9.76 microgram/ml (29.3 microM) and a maximum velocity of 59.3 ng/min/10(6) cells. Transport by both pathways was Na(+) independent. Ciprofloxacin transport by quiescent cells was relatively insensitive to pH and N-ethylmaleimide but was competitively inhibited by adenine (K(i) = 1.55 mM). Papaverine, a benzylisoquinoline known to inhibit nucleobase transport, also inhibited ciprofloxacin transport by quiescent cells. In contrast, transport by PMA-stimulated cells was enhanced at pH 8.2, inhibited at pH 6.2, and blocked by N-ethylmaleimide. Cationic and neutral amino acids and cystine competitively inhibited ciprofloxacin transport by PMA-stimulated neutrophils (K(i) = 158 microM for ornithine) but had little effect on quiescent cells. PMA-activated transport was not inhibited when the Na(+) in the medium was replaced with K(+) or Li(+), and the pattern of inhibition by cationic and neutral amino acids was similar. In summary, neutrophils continuously transport ciprofloxacin via a transport pathway shared by adenine. Activation by PMA induces a separate, higher-affinity transport pathway shared by a broad scope of amino acids. Neutrophils utilize one or both of these mechanisms to transport other fluoroquinolones.

Modulation of Ca2+ exchange with the Ca(2+)-specific regulatory sites of troponin C.

Calcium (Ca2+) binding to the N-terminal Ca(2+)-specific sites on troponin C (TnC) regulate the contraction-relaxation cycle of skeletal muscle. A mutant TnC (F29W) and dansylaziridine-labeled TnC undergo large fluorescence increases when Ca2+ binds to their Ca(2+)-specific sites (half-maximal at pCa 5.8). Calmidazolium and the additional mutation of Met-82 to Gln (F29W,M82Q) increased Ca2+ affinity at these Ca2+ sites by approximately 4-fold (half-maximal at pCa approximately 6.4). Calmidazolium and the M82Q mutation decreased the rate of Ca2+ dissociation from the Ca(2+)-specific sites approximately 3.4-fold (from approximately 462 +/- 84/s to approximately 138 +/- 30/s) at 22 degrees C. Ca2+ associated with the Ca(2+)-specific sites of these proteins at 1-2 x 10(8) M-1 s-1 at 4 degrees C. These drug- and mutation-induced increases in Ca2+ affinity occur solely from large decreases in the Ca2+ off-rate without an effect on the Ca2+ on-rate. Thus, Ca2+ can bind to the Ca(2+)-specific sites of TnC as rapidly as it can diffuse to the protein, consistent with the extreme speed of skeletal muscle contraction. Drugs and/or site-directed mutagenesis can modify the Ca2+ sensitivity and the rate of Ca2+ exchange with TnC's Ca(2+)-specific sites to perhaps alter the rate of relaxation and/or the rate of rise of tension.