Porphyromonas gingivalis reduces mitogenic and chemotactic responses of human periodontal ligament cells to platelet-derived growth factor in vitro.

The effects of a sonicated Porphyromonas gingivalis ATCC 33277 protein extract on the mitogenic and chemotactic responses of human periodontal ligament (PDL) cells to the recombinant human platelet-derived growth factor-BB homodimer (PDGF-BB) were examined in vitro. Proliferation of PDL cells was inhibited by P. gingivalis extract at concentrations higher than 10 micrograms/mL protein. At 100 micrograms/mL of P. gingivalis extract, cells did not proliferate. DNA synthesis in PDL cells, as revealed by [3H]-thymidine incorporation, was also inhibited by approximately 50% in the presence of 50 micrograms/mL P. gingivalis extract for 24 hours. In contrast, PDGF-BB at 1 ng/mL enhanced DNA synthesis in PDL cells, followed by maximum enhancement at concentrations higher than 10 ng/mL PDGF-BB. However, this mitogenic response to PDGF-BB was markedly reduced in the presence of 20 micrograms/mL of P. gingivalis extract and did not reach the maximum level even if PDGF-BB concentrations were increased to 250 ng/mL. PDL cells exhibited a chemotactic response to PDGF-BB at 1 ng/mL, which was also inhibited by pretreatment of the cells with P. gingivalis extract at 10 to 50 micrograms/mL. Scatchard analysis of a [125I]-PDGF binding assay demonstrated that PDL cells have both high and low PDGF binding affinity sites. Treatment of the cells with P. gingivalis extract decreased the number of PDGF-binding sites to approximately 35% of the control level, while it caused only a slight change in the affinities of both types of binding site. These results indicated that the P. gingivalis extract reduced mitogenic and chemotactic responses of human PDL cells, possibly through mechanisms involving a decrease in PDGF-binding capacity of these cells. Due to this inhibitory effect of P. gingivalis, the normal levels of PDGF in periodontal lesions may not be sufficient to promote periodontal regeneration through activation of PDL cell proliferation and migration. Therefore, the therapeutic use of PDGF-BB, as a supplement to pre-existing PDGF and as an adjunct, while also eliminating P. gingivalis from periodontal lesions, would help periodontal tissue regeneration.

Mitogenic, chemotactic, and synthetic responses of rat periodontal ligament fibroblastic cells to polypeptide growth factors in vitro.

The mitogenic, chemotactic, and synthetic responses of rat periodontal ligament (PDL) fibroblastic cells to epidermal growth factor (EGF), transforming growth factor-beta (TGF-beta), recombinant human platelet-derived growth factor (rhPDGF)-AB, rhPDGF-BB, natural (n) PDGF-AB, and insulin-like growth factor-I (IGF-I) were examined in vitro using PDL cells obtained from the coagulum of healing tooth sockets. PDGFs and IGF-I have potent and comparable mitogenic effects on PDL fibroblastic cells. The maximum mitogenic effect of PDGFs was observed at the concentration of 10 ng/ml, whereas that of IGF-I was seen at concentrations higher than 100 ng/ml. In contrast, EGF induced moderate, and TGF-beta inhibitory mitogenic responses. The combination of rhPDGF-AB with either EGF or TGF-beta demonstrated comparable mitogenic potency, equivalent to the level of PDGF alone regardless of the mitogenic effect of other growth factors. The combination of rhPDGF-AB and IGF-I, however, showed a synergistic effect revealing the highest mitogenic effect among all individual growth factors as well as any combinations of the growth factors tested. Similarly, PDL fibroblastic cells demonstrated strong chemotactic responses to both IGF-I and PDGFs. The maximum effect was observed by IGF-I at concentrations higher than 10 ng/ml, followed by rhPDGF-BB at 0.1 ng/ml, rhPDGF-AB and nPDGF at concentrations ranging from 0.1 to 1 ng/ml. TGF-beta revealed no, and EGF slightly increased, chemotactic effects. IGF-I slightly enhanced the synthesis of total protein, whereas other factors had no significant effect. However, both rhPDGF-AB and TGF-beta stimulated collagen synthesis. On the other hand, IGF-I showed no effect on collagen synthesis, while EGF suppressed collagen synthesis. These findings suggest that rhPDGF-BB and IGF-I stimulate proliferation and chemotaxis of PDL fibroblastic cells. In addition, the combination of these growth factors further increases the mitogenic effect. rhPDGF-AB also stimulates collagen synthesis by PDL fibroblastic cells. Thus, rhPDGF-BB and IGF-I may have important roles in promotion of PDL healing, and consequently, may be useful for clinical application in periodontal regenerative procedures.

Role of epidermal growth factor and its receptor in mechanical stress-induced differentiation of human periodontal ligament cells in vitro.

The periodontal ligament (PDL) contains precursor cells for osteoblasts and cementoblasts. It has been shown that epidermal growth factor (EGF) inhibits dexamethasone-induced differentiation and up-regulates EGF-receptor (EGF-R) expression, whereas EGF-R is down-regulated in the course of differentiation. Thus it was suggested that EGF and its receptors act as a negative regulator of osteoblastic differentiation in PDL cells. In order to investigate further this hypothesis, human PDL cells were now used to elucidate the role of EGF and EGF-R in their proliferation and differentiation under mechanical stress-loaded conditions in vitro, as the PDL regularly receives mechanical stress from occlusal forces. As a model of mechanical stress, a cyclic stretch of 9 or 18% elongation was applied to the cells with a Flexercell cell-strain unit system. Alkaline phosphatase activity and osteocalcin mRNA expression were significantly induced by loading cyclic stretch for more than 4 days, whereas stretch slightly inhibited cell proliferation. Visualization of the actin stress fibres of the cells by rhodamine phalloidin revealed that approx. 10% of the total number of cells had become aligned perpendicularly to the direction of the stretch. The effects of stretch on alkaline phosphatase activity and cell proliferation were totally abolished by the presence of 10 ng/ml EGF. Western blotting of EGF-R protein demonstrated that stretch-induced differentiation accompanied the decreased expression of EGF-R protein in the cells. However, the amount of tyrosine-phosphorylated EGF-R upon EGF stimulation was restored to the control level in stretched cells. These results suggest that the EGF/EGF-R system acts as a negative regulator of differentiation of PDL cells regardless of the type of differentiation stimuli. Also, interaction between mechanical stress and the EGF/EGF-R system may participate in the osteoblastic differentiation of PDL cells and thereby regulate the source of cementoblasts and osteoblasts.

Technical evaluation of oxygen transfer rates of fish gills and artificial gills.

An artificial gill, which takes oxygen from water, would enhance the ability of people to function under water for extended periods. Increasing oxygen transfer rate, however, would be essential to the realization of compact, commercially viable equipment. Fish have evolved a variety of techniques to enable them to breathe under water, and their mechanisms must be clarified before compact, high-performance artificial gills can be developed. A model of the secondary lamellae of fish gills, through which oxygen is taken up from water to the blood, was devised, and its structure and oxygen transfer rate were evaluated by computer simulation analysis for carp and dogfish. Oxygen transfer rates were also found for an outside-water-flow artificial gill using a hollow fiber membrane at various fiber packing ratios. The biologic membrane is rate-determining for oxygen transfer through the secondary lamellae. Blood and water side boundary film resistances are small for fish because the blood and water channels are very narrow and numerous. When the fiber packing ratio of the artificial gill is raised, the oxygen transfer rate increases because of lower water side boundary film resistance. An optimum fiber packing ratio should be selected so that there is no major increase in pressure drop and no channeling occurs.

Microbial contamination of a disinfectant-soaked unwoven cleaning cloth.

In December 2009, a 76-year-old male patient developed pneumonia due to Burkholderia cepacia whilst in an intensive care unit at a Japanese university hospital. During the subsequent environmental investigation to find the source, B. cepacia with an identical DNA type was found in his denture storage solution. Open packets of unwoven rayon cloths soaked in 0.2% alkyldiaminoethylglycine hydrochloride, used for environmental cleaning, were shown to be contaminated with B. cepacia, Alcaligenes xylosoxidans, Pseudomonas fluorescens and Pseudomonas aeruginosa. B. cepacia of a different DNA type was found in five of 42 samples from sealed packets of cloths.

Permeability properties of gels and membranes derived from chitosan.

A series of membranes was prepared by air-drying the thin layers of N-acyl- and N-arylidene-chitosan gels. Their flow rates of water and permeabilities of various compounds were examined. N-Acylchitosan membranes were stable in both dilute acid and alkali, but N-arylidene-chitosan membranes were unstable in dilute acid. N-Acetylchitosan membranes were stable in formic acid at room temperature for up to 7 hr. The flow rates of water through N-acetylchitosan membranes were 10.0--23.6 X 10(-3) ml/cm2min under a pressure of 3 kg/cm2, and were unchanged by the membrane thickness (12--60 micrometers). The increase of carbon numbers for N-acyl groups caused a slight decrease in the flow rates, and the flow rates were decreased by partial O-acetylation of N-acetylchitosan membranes. The flow rate of water through chitosan membranes (thickness 30--35 micrometers) was 7.1 X 10(-4) ml/cm2min, which was decreased by an increase in the membranes thickness. Low-molecular-weight compounds (MW less than 2900) passed through these membranes, but high molecular-weight compounds (MW greater than 13,000) did not pass through.

Healing of segmental bone defects in rats induced by a beta-TCP-MCPM cement combined with rhBMP-2.

A beta-tricalcium phosphate-monocalcium phosphate monohydrate (beta-TCP-MCPM) cement was evaluated as an effective carrier of recombinant human bone morphogenetic protein-2 (rhBMP-2) in rat femoral critical-size defects. Hard cement cylinders (4 x 5 mm) impregnated with two different doses of rhBMP-2 (1.26 or 6.28 microg) were implanted into each defect, and the results were compared with those in rats that had implantations of cylinders only. Implantation of the 6.28 microg dose of rhBMP-2 caused a large bone shell to form around the defect, resulting in osseous union in all cases within 3 weeks. Except for beta-TCP granules, the cement was resorbed and replaced by bone tissue at 6 weeks. A torsion test at 9 weeks showed that the failure torque and bone stiffness had recovered 99% and 141%, respectively, compared with the intact contralateral femur. The defects that received 1.26 microg of rhBMP-2 resulted in 40% union and 41% of the failure torque at 9 weeks. However, no instances of union were observed in the defects implanted with cylinders only. In conclusion, the beta-TCP-MCPM cement was shown to be effective as a rhBMP-2 carrier. Combined with rhBMP-2, this cement was rapidly resorbed and completely healed the defects.

Porphyromonas gingivalis lipopolysaccharide modulates the responsiveness of human periodontal ligament fibroblasts to platelet-derived growth factor.

Lipopolysaccharides (LPS) prepared from periodontopathic bacteria have been known to induce various biological responses which may lead to periodontal tissue breakdown. The purpose of this study was to determine if Porphyromonas gingivalis LPS could affect cellular functions of human periodontal ligament fibroblasts (HPLF). We showed here the responsiveness of cultured HPLF to platelet-derived growth factor (PDGF)-BB, a growth factor for mesenchymal cells, in the presence of P. gingivalis LPS. DNA synthesis of HPLF was enhanced in a dose-dependent manner when LPS were co-incubated for 48 h; thereafter, it decreased to the baseline level within 24 h incubation. The stimulating effect of PDGF-BB was further enhanced by the pretreatment of HPLF with LPS (10 micrograms/ml) for 48 h. The binding assay of [125I]PDGF-BB and the flow cytometric assay using rabbit antiserum to human PDGF receptor (PDGF-R) beta-type indicated that this enhancement was due to the increase of the number of PDGF-R beta-type on HPLF. Immunoprecipitation using antiserum to human PDGF-R beta-type also showed that the synthesis of PDGF-R beta-type was augmented in the LPS-treated HPLF. These results indicate that P. gingivalis LPS stimulate cellular proliferation and responsiveness to PDGF-BB of cultured HPLF. These cellular reactions may be mediated by PDGF-BB binding, followed by increased synthesis of the receptor protein.

In vitro formation of mineralized nodules by periodontal ligament cells from the rat.

The purposes of this study were to determine whether periodontal ligament (PDL) cells are capable of producing mineralized nodules in vitro and to analyze ultrastructural features of the nodules. Rat PDL cells were obtained from coagulum in the socket at 2 days after tooth extraction and cultured at confluence in standard medium containing Dulbecco's Modified Eagle's Medium supplemented with 10% FBS and antibiotics. To test mineralized nodule formation, cells were further cultured for an additional 3 weeks in the standard medium containing (1) ascorbic acid (50 micrograms/ml) and sodium beta-glycerophosphate (10 mM), (2) ascorbic acid, sodium beta-glycerophosphate, and dexamethasone (5 microM), or (3) ascorbic acid alone. Cells were then fixed in 2.5% glutaraldehyde, postfixed in 1% OsO4, and prepared for light and electron microscopy. Three-dimensional nodules containing mineralized matrices were formed only when the cells were cultured in the presence of ascorbic acid and dexamethasone. They were composed of multilayered fibroblasts (up to 13 layers), and highly organized collagen fibrils with 64 nm cross-banding patterns between the cell layers. The fibroblasts in the nodules exhibited an elongated shape with a high degree of cytoplasmic polarity throughout the nodule, and have the morphological features of PDL fibroblasts as seen in vivo. Mineral deposition with needle-like crystals was initiated on collagen fibrils located in intercellular spaces of the upper cell layers and became increasingly heavier towards the bottom half of the nodules. X-ray microanalysis and electron diffraction analysis confirmed that mineral deposition contained calcium and phosphate in the form of immature hydroxyapatite.(ABSTRACT TRUNCATED AT 250 WORDS)

Contact sensitivity to polychloroparaxylene-coated cardiac pacemaker.

Polychloroparaxylene (parylene) is widely used as a material for cardiac pacemaker coating. Contact sensitivity to parylene was proven by patch test. Wrapping the pacemaker in a polytetrafluoroethylene sheet prior to implantation prevented further skin reactions.

Predominant contribution of the G protein-mediated mechanism to NaF-induced vascular contractions in diabetic rats: association with an increased level of G(qalpha) expression.

The purpose of this study was to determine the mechanism responsible for alterations in NaF-induced contractions of blood vessels from streptozotocin-induced diabetic rats. In the presence of AlCl(3), NaF (>/=7.5 mM) produced significantly greater contractions in diabetic aorta and mesenteric artery compared with age-matched controls. Pretreatment with 1 microM nifedipine eliminated the enhanced contractile responses of diabetic vessels to NaF, resulting in no difference in the magnitude of NaF-induced contractions between control and diabetic vessels. In the presence of 100 microM deferoxamine, an Al(3+) chelator, NaF-induced contractions of diabetic vessels were markedly attenuated, whereas only the responses to lower concentrations of NaF were reduced in control vessels. No significant difference was found in the peak amplitude of transient contractions induced by 10 microM cyclopiazonic acid between control and diabetic vessels. The addition of 10 microM okadaic acid produced attenuated contractions in diabetic vessels. These findings indicate no involvement of the inhibitory effects of NaF on endoplasmic reticular Ca(2+)-pump ATPase and protein phosphatases in the genesis of the enhanced responsiveness of diabetic vessels to NaF. Western blot analysis showed a 2.5-fold increase in the expression of G(qalpha) in diabetic aortic membranes. In contrast, the G(ialpha) level was modestly decreased and the G(salpha) and G(betagamma) levels were unchanged in diabetes. The present results suggest that enhanced vascular contractions to NaF in diabetes is attributed predominantly to a G protein-mediated Ca(2+) channel activation that results from markedly increased G(qalpha) expression in vascular tissues under this pathological state.

Acellular and cellular hemoglobin solutions as vasoconstrictive factor.

The inhibitory effects of acellular and cellular hemoglobin (Hb) solutions on endothelium-dependent vasorelaxation were investigated in rabbit thoracic aortic strips. As acellular Hb solutions, 2,3-diphosphoglycerate (DPG)-depleted Hb and pyridoxylated Hb were examined. Cellular Hb solutions included washed human fresh red cells and liposome Hb encapsulated with pyridoxal-5'-phosphate (PLP). The tissues were precontracted with phenylephrine (PE), after which acetylcholine (ACh) was added to elicit a steady-state relaxation. Acellular Hb solutions cumulatively reversed ACh-induced relaxation, and these inhibitory effects reached a plateau at 10 micrograms/ml. Increasing oxygen affinity by pyridoxylation had little effect on this. In contrast, both red cells and liposome Hb solution showed moderate inhibitory effects, and they reached a plateau at 1 mg/ml. These findings indicate that acellular Hb solutions are more potent inhibitors than cellular Hb solutions by a factor of about 100, and that the encapsulation of Hb is a preferable method to mimic the red cell.

A sensitive enzymatic method (SK-013) for detection and quantification of specific periodontopathogens.

Porphyromonas gingivalis, Bacteroides forsythus, and Treponema denticola have been found to predominate in periodontal pockets of patients with adult periodontitis. These microorganisms hydrolyze the synthetic peptide N-benzoyl-DL-arginine-2-naphthylamide (BANA). In this study, we developed an enzymatic method, designated SK-013, to detect the existence of these microorganisms in subgingival plaque bacteria. This enzymatic method was based on the observation of the hydrolysis of N-carbobenzoxy-glycyl-glycyl-arginyl-3,5-dibromo-4-hydroxyaniline (N-CBz-Gly-Gly-Arg-DBHA) and made more sensitive by adding an enhancing system. The SK-013 was specifically positive for P. gingivalis, B. forsythus, T. denticola, and some strains of Capnocytophaga species, but was not specific for any of the other bacterial strains tested. This SK-013 system may be valuable for detection and quantification of periodontal disease-associated bacteria in subgingival plaque and thus for diagnosis of periodontal infections.

[A rapid diagnosis (SK-013) for periodontitis based on the enzymatic activity of periodontopathic bacteria. Study on the characterization of the SK-013 and the enzyme specificity].

We present studies for development of an enzymatic diagnostic method for periodontitis. A rapid and sensitive diagnostic method named SK-013 was provided by Sunstar Inc. to evaluate the peptidase activities specifically derived from periodontopathic bacteria such as Bacteroides gingivalis, Bacteroides forsythus and Treponema denticola and some strains of Capnocytophaga species. The results obtained indicated the specificity of the enzymatic reaction in this system.