Resistance to vertical root fracture of root filled teeth using different conceptual approaches to canal preparation.

AIM: To evaluate the resistance to vertical root fracture of root filled teeth using different root canal preparation concepts: apical stop and continuously tapering preparation, with different foraminal enlargement. In addition, the effect of filling associated with these different concepts was assessed. METHODOLOGY: Ninety single-rooted premolars were used. The crowns were removed to standardize the root length at 11 mm. Ten roots were not instrumented and not filled (control group). The other roots were divided into four groups (n = 20): apical stop to finishing file size 25, 0.08 taper; apical stop to finishing file size 45, 0.02 taper; continuously tapering preparation to finishing file size 25, 0.08 taper; and continuously tapering preparation to finishing file size 45, 0.02 taper. Afterwards, 10 roots of each group were root filled using Gutta-percha and AH Plus. A continuous wave of condensation was used as the filling technique. The roots were evaluated as regards resistance to vertical fracture using a universal testing machine. The data were evaluated using the Kruskal-Wallis and Mann-Whitney tests. RESULTS: No significant difference in performance was observed between continuously tapering preparation size 25, 0.08 taper and apical stop size 45, 0.02 taper groups. Lower resistance values were found in continuously tapering preparation size 45, 0.02 taper group and the highest in apical stop size 25, 0.08 taper group. After filling, a significant increase in resistance values was observed in all groups. In addition, the continuously tapering preparation size 45, 0.02 taper group had values comparable with those of the control. CONCLUSION: The different canal preparation techniques reduced resistance to fracture when compared with the control group; however, after root filling, there was a significant increase in resistance.

Ex vivo antimicrobial efficacy of the EndoVac system plus photodynamic therapy associated with calcium hydroxide against intracanal Enterococcus faecalis.

AIM: To evaluate the ex vivo efficacy of the EndoVac system and photodynamic treatment (PDT) as adjuncts to chemomechanical debridement associated with calcium hydroxide (CaOH2 ) in reducing the levels of intracanal Enterococcus faecalis. METHODOLOGY: One hundred and twenty-five sterile premolar teeth were conventionally accessed, prepared and then contaminated with E. faecalis (ATCC 29212) for 30 days. Teeth were randomly divided into 4 groups: Control (chemomechanical debridement with conventional irrigation); Endovac (chemomechanical debridement with EndoVac system); PDT (chemomechanical debridement with conventional irrigation and PDT) and Endovac+PDT (chemomechanical debridement with EndoVac and PDT). The irrigants used in all groups were 5.25% sodium hypochlorite and 17% EDTA. After treatment, an intracanal dressing (CaOH2 ) was applied in all canals for 7 days. Samples were obtained before (T1) and after the therapeutic procedures (T2) and, after intracanal medication (T3), plated onto BHI media and incubated (37 °C, 48 h) to determine the colony-forming units (CFU mL(-1) ). RESULTS: The overall mean cell counts (CFU mL(-1) ) of E. faecalis were high at the initial contamination (T1). A significant reduction (P < 0.05) of E. faecalis mean counts was observed in all groups from baseline (T1) to both post-therapy samplings (T2 and T3); no differences amongst the groups were detected. No significant change in bacterial counts from T2 to T3 was detected. CONCLUSION: The adjunctive use of the EndoVac system and the photodynamic treatment, in combination or not, was as effective as the conventional chemomechanical debridement associated with CaOH2 on reducing the counts of intracanal E. faecalis.

Is salivary histatin 5 a metallopeptide?

Histatins are small histidine-rich salivary polypeptides which exhibit antimicrobial activity against Candida albicans. This antimicrobial activity has been ascribed in part to a high content of basic amino acids. However, unlike most other antimicrobial proteins histatins have a high content of histidine, tyrosine and acidic amino acids known to participate in metal ion coordination. This study was conducted to test whether histatin 5 could bind zinc and copper which are metals present in salivary secretions and whole saliva. Physical binding parameters and spectral properties of zinc- and copper-histatin complexes were investigated in order to obtain direct evidence of these interactions. A spectrophotometric competition assay using the metallochromic indicator murexide showed that histatin 5 dissociates metal indicator complexes containing zinc or copper ions. Absorption spectra of histatin 5 at increasing copper chloride concentrations resulted in higher absorbance in the 230-280 nm wavelength range and this spectral change was saturated at a peptide:metal molar ratio of approx. 1:1. A corresponding band was observed in the visible range of the spectrum with a maximum and molar extinction coefficient corresponding to that of copper binding to an ATCUN motif. Quantitative assessment of zinc and copper binding to histatin 5 using isothermal titration calorimetry revealed at least one high affinity site for each metal, with binding constants of 1.2x10(5) and 2.6x10(7) M(-1), respectively. These results indicate that histatin 5 exhibits metallopeptide-like properties. The precise biological significance of this has not yet been established but histatins may contribute significantly to salivary metal binding capacity.

Salivary histatin 5 is a potent competitive inhibitor of the cysteine proteinase clostripain.

Histatin 5 is a low molecular weight salivary protein which is known to exhibit inhibitory activity against several proteinases, including the cysteine proteinases gingipains. The purpose of this study was to characterize the effect of salivary histatin on the proteolytic activity of the cysteine proteinase clostripain derived from the pathogen Clostridium histolyticum. Using a synthetic nitroanilide substrate, we studied in detail the inhibition of clostripain by histatin 5 and compared the effect of this peptide to that of leupeptin, a known competitive inhibitor of clostripain. It was found that the concentration of histatin 5 required to inhibit 50% of clostripain activity was 23.6+/-1.6 nM. Kinetic analysis revealed that histatin 5 is a competitive inhibitor of clostripain with an inhibition constant (K(i)) of 10 nM. The K(i) for the inhibition of clostripain activity against nitroanilide substrate by leupeptin was found to be 60 nM, significantly higher than that of histatin 5. Thus, histatin 5 inhibits clostripain more effectively than leupeptin and other cysteine protease inhibitors studied here. No significant proteolysis of histatin 5 was observed when histatin 5 was incubated at physiologic concentrations with clostripain. The potent inhibition of clostripain by histatin 5 points towards the possibility that this protein may prevent establishment of clostridial infections and therefore may have significant potential for the treatment of diseases associated with this enzyme.

The response of human buccal maxillary furcation defects to combined regenerative techniques--two controlled clinical studies.

Treatment of maxillary furcation lesions presents important clinical challenges. The aim of this study was to evaluate the response of buccal class II furcation defects in systemically healthy, non-smoking, Adult Periodontitis patients undergoing supportive periodontal therapy and surgical periodontal regenerative techniques. The treatment protocol emphasised infection control, careful soft tissue management and wound stability. Two experiments were performed. In the first experiment, a coronally positioned flap (CPF) technique was compared with surgical debridement. In the second experiment, an experimental technique (ET) was also compared with surgical debridement. The ET was a combination of a composite graft consisting of microparticulate (40-70 microns) hydroxyapatite and tetracycline (3:1), a guided tissue regeneration barrier (GTR) and CPF. Sixty patients were divided into the four (n = 15) treatment groups. The clinical variables evaluated were plaque, bleeding-on-probing scores, gingival recession, probing pocket depth (PD), vertical (VAL) and horizontal attachment levels (HAL). Reevaluation was performed 12 months after the surgical procedures and revealed that the three techniques resulted in improvements in all the clinical variables evaluated. Post-operative measurements from controls, CPF and ET showed (respectively) 1.53 +/- 1.12 mm, 1.40 +/- 0.75 mm, 2.43 +/- 1.36 mm reduction in PD; 0.63 +/- 0.1 mm, 1.17 +/- 1.12 mm and 1.57 +/- 1.32 mm VAL gains and 1.03 +/- 0.30 mm, 1.40 +/- 0.96 mm and 2.13 +/- 1.52 mm HAL gains. Five out of fifteen furcations in the ET group were closed after treatment. It was concluded that satisfactory clinical results--measured by PD reduction, VAL and HAL gains--can be obtained in advanced defects formerly considered as being non-responsive to regenerative periodontal therapy. The proposed ET showed significantly better results, with more PD reduction, HAL and VAL gains and a higher frequency of furcation closure compared with the other techniques tested, and shows promise as a new regenerative treatment technique.

A new method for the isolation of histatins 1, 3, and 5 from parotid secretion using zinc precipitation.

Histatins, a family of small-molecular-weight, histidine-rich cationic salivary proteins, have been difficult to isolate in an efficient way by conventional procedures due to their anomalous interactions with chromatographic resins. In the present study we explored the possibility of developing a new isolation procedure based on recent observations that histatins associate with various metal ions, including zinc. Since solubility studies showed that histatin 5 forms precipitates with zinc under alkaline conditions, we investigated whether this characteristic could be exploited for the preparative isolation of histatins from salivary secretions. A fast and efficient two-step procedure was developed using zinc precipitation of histatins from human parotid secretion followed by final purification using reversed-phase high-performance liquid chromatography (HPLC). Analysis of zinc precipitates by Tricine-SDS-PAGE, cationic PAGE, HPLC, and mass spectrometry revealed the presence of the three major histatins, 1, 3, and 5, as well as statherin. The histatin yield obtained by the precipitation step was approximately 90%. Therefore, zinc precipitation of histatins from glandular salivary secretions is a novel, rapid, and effective means for the isolation of these proteins.

Salivary histatin 5 is an inhibitor of both host and bacterial enzymes implicated in periodontal disease.

One of the salient features of periodontitis and gingivitis is the increase in the levels of bacterial and host-derived proteolytic enzymes in oral inflammatory exudates. This study evaluated the potential of histatin 5, a 24-residue histidine-rich salivary antimicrobial protein, to inhibit these enzymes. Using biotinylated gelatin as a substrate, histatin 5 was found to inhibit the activity of the host matrix metalloproteinases MMP-2 and MMP-9 with 50% inhibitory concentrations (IC50s) of 0.57 and 0.25 microM, respectively. To localize the domain responsible for this inhibition, three peptides containing different regions of histatin 5 were synthesized and tested as inhibitors of MMP-9. Peptides comprising residues 1 to 14 and residues 4 to 15 of histatin 5 showed much lower inhibitory activities (IC50, 21.4 and 20.5 microM, respectively), while a peptide comprising residues 9 to 22 showed identical activity to histatin 5 against MMP-9. These results point to a functional domain localized in the C-terminal part of histatin 5. To evaluate the effect of histatin 5 on bacterial proteases, a detailed characterization of histatin 5 inhibition of gingipains from Porphyromonas gingivalis was carried out using purified Arg- and Lys-specific enzymes. Kinetic analysis of the inhibition of the Arg-gingipain revealed that histatin 5 is a competitive inhibitor, affecting only the Km with a K(i) of 15 microM. In contrast, inhibition of Lys-gingipain affected both the Km and Vmax, suggesting that both competitive and noncompetitive competitive processes underlie this inhibition. The inhibitory activity of histatin 5 against host and bacterial proteases at physiological concentrations points to a new potential biological function of histatin in the oral cavity.