Endodontic biofilms: contemporary and future treatment options

Apical periodontitis is a biofilm-mediated infection. The biofilm protects bacteria from host defenses and increase their resistance to intracanal disinfecting protocols. Understanding the virulence of these endodontic microbiota within biofilm is essential for the development of novel therapeutic procedures for intracanal disinfection. Both the disruption of biofilms and the killing of their bacteria are necessary to effectively treat apical periodontitis. Accordingly, a review of endodontic biofilm types, antimicrobial resistance mechanisms, and current and future therapeutic procedures for endodontic biofilm is provided.

Analysis of temperature changes and sterilization effect of diode laser for the treatment of peri-implantitis by wavelength and irradiation time

PURPOSE: We compared the effects of newly developed diode laser (Bison 808 nm Diode laser) on the treatment of peri-implantitis with conventional products (Picasso 810 nm Diode laser) by comparing the surface temperature of titanium disc and bacterial sterilization according to laser power. MATERIALS AND METHODS: The titanium disc was irradiated for 60 seconds and 1 – 2.5 W using diode laser 808 nm and 810 nm. The surface temperature of the titanium disc was measured using a temperature measurement module and a temperature measurement program. In addition, in order to investigate the sterilizing effect according to the laser power, 808 nm laser was irradiated after application of bacteria to sandblasted large-grit acid-etched (SLA) and resorbable blast media (RBM) coated titanium discs. The irradiated disks were examined with scanning electron microscopy. RESULTS: Both 808 nm and 810 nm lasers increased disk surface temperature as the power increased. When the 810 nm was irradiated under all conditions, the initial temperature rise rate, the descending rate, and the temperature change before and after was higher than that of 808 nm. Disk surface changes were not observed on both lasers at all conditions. Bacteria were irradiated with 808 nm, and the bactericidal effect was increased as the power increased. CONCLUSION: When applying these diode lasers to the treatment of peri-implantitis, 808 nm which have a bactericidal effect with less temperature fluctuation in the same power conditions would be considered safer. However, in order to apply a laser treatment in the dental clinical field, various safety and reliability should be secured.

Antifungal effects of synthetic human β-defensin 3-C15 peptide

OBJECTIVES: The purpose of this ex vivo study was to compare the antifungal activity of a synthetic peptide consisting of 15 amino acids at the C-terminus of human β-defensin 3 (HBD3-C15) with calcium hydroxide (CH) and Nystatin (Nys) against Candida albicans (C. albicans) biofilm. MATERIALS AND METHODS: C. albicans were grown on cover glass bottom dishes or human dentin disks for 48 hr, and then treated with HBD3-C15 (0, 12.5, 25, 50, 100, 150, 200, and 300 µg/mL), CH (100 µg/mL), and Nys (20 µg/mL) for 7 days at 37℃. On cover glass, live and dead cells in the biomass were measured by the FilmTracer Biofilm viability assay, and observed by confocal laser scanning microscopy (CLSM). On dentin, normal, diminished and ruptured cells were observed by field-emission scanning electron microscopy (FE-SEM). The results were subjected to a two-tailed t-test, a one way analysis variance and a post hoc test at a significance level of p = 0.05. RESULTS: C. albicans survival on dentin was inhibited by HBD3-C15 in a dose-dependent manner. There were fewer aggregations of C. albicans in the groups of Nys and HBD3-C15 (≥ 100 µg/mL). CLSM showed C. albicans survival was reduced by HBD3-C15 in a dose dependent manner. Nys and HBD3-C15 (≥ 100 µg/mL) showed significant fungicidal activity compared to CH group (p < 0.05). CONCLUSIONS: Synthetic HBD3-C15 peptide (≥ 100 µg/mL) and Nys exhibited significantly higher antifungal activity than CH against C. albicans by inhibiting cell survival and biofilm.

Effect of acidic solutions on the microhardness of dentin and set OrthoMTA and their cytotoxicity on murine macrophage

OBJECTIVES: To evaluate the effects of three acids on the microhardness of set mineral trioxide aggregate (MTA) and root dentin, and cytotoxicity on murine macrophage. MATERIALS AND METHODS: OrthoMTA (BioMTA) was mixed and packed into the human root dentin blocks of 1.5 mm diameter and 5 mm height. Four groups, each of ten roots, were exposed to 10% citric acid (CA), 5% glycolic acid (GA), 17% ethylenediaminetetraacetic acid (EDTA), and saline for five minutes after setting of the OrthoMTA. Vickers surface microhardness of set MTA and dentin was measured before and after exposure to solutions, and compared between groups using one-way ANOVA with Tukey test. The microhardness value of each group was analyzed using student t test. Acid-treated OrthoMTA and dentin was examined by scanning electron microscope (SEM). Cell viability of tested solutions was assessed using WST-8 assay and murine macrophage. RESULTS: Three test solutions reduced microhardness of dentin. 17% EDTA demonstrated severe dentinal erosion, significantly reduced the dentinal microhardness compared to 10% CA (p = 0.034) or 5% GA (p = 0.006). 10% CA or 5% GA significantly reduced the surface microhardness of set MTA compared to 17% EDTA and saline (p < 0.001). Acid-treated OrthoMTA demonstrated microporous structure with destruction of globular crystal. EDTA exhibited significantly more cellular toxicity than the other acidic solutions at diluted concentrations (0.2, 0.5, 1.0%). CONCLUSIONS: Tested acidic solutions reduced microhardness of root dentin. Five minute's application of 10% CA and 5% GA significantly reduced the microhardness of set OrthoMTA with lower cellular cytotoxicity compared to 17% EDTA.

Effect of acidic solutions on the microhardness of dentin and set OrthoMTA and their cytotoxicity on murine macrophage

OBJECTIVES: To evaluate the effects of three acids on the microhardness of set mineral trioxide aggregate (MTA) and root dentin, and cytotoxicity on murine macrophage. MATERIALS AND METHODS: OrthoMTA (BioMTA) was mixed and packed into the human root dentin blocks of 1.5 mm diameter and 5 mm height. Four groups, each of ten roots, were exposed to 10% citric acid (CA), 5% glycolic acid (GA), 17% ethylenediaminetetraacetic acid (EDTA), and saline for five minutes after setting of the OrthoMTA. Vickers surface microhardness of set MTA and dentin was measured before and after exposure to solutions, and compared between groups using one-way ANOVA with Tukey test. The microhardness value of each group was analyzed using student t test. Acid-treated OrthoMTA and dentin was examined by scanning electron microscope (SEM). Cell viability of tested solutions was assessed using WST-8 assay and murine macrophage. RESULTS: Three test solutions reduced microhardness of dentin. 17% EDTA demonstrated severe dentinal erosion, significantly reduced the dentinal microhardness compared to 10% CA (p = 0.034) or 5% GA (p = 0.006). 10% CA or 5% GA significantly reduced the surface microhardness of set MTA compared to 17% EDTA and saline (p < 0.001). Acid-treated OrthoMTA demonstrated microporous structure with destruction of globular crystal. EDTA exhibited significantly more cellular toxicity than the other acidic solutions at diluted concentrations (0.2, 0.5, 1.0%). CONCLUSIONS: Tested acidic solutions reduced microhardness of root dentin. Five minute's application of 10% CA and 5% GA significantly reduced the microhardness of set OrthoMTA with lower cellular cytotoxicity compared to 17% EDTA.

Comparison of the rheological properties of four root canal sealers / 国际口腔科学杂志·英文版

The flowability of a root canal sealer is clinically important because it improves the penetration of the sealer into the complex root canal system. The purpose of this study was to compare the flowabilities of four root canal sealers, measured using the simple press method (ISO 6876), and their viscosities, measured using a strain-controlled rheometer. A newly developed, calcium phosphate-based root canal sealer (Capseal) and three commercial root canal sealers (AH Plus, Sealapex and Pulp Canal Sealer EWT) were used in this study. The flowabilities of the four root canal sealers were measured using the simple press method (n=5) and their viscosities were measured using a strain-controlled rheometer (n=5). The correlation between these two values was statistically analysed using Spearman's correlation test. The flow diameters and the viscosities of the root canal sealers were strongly negatively correlated (ρ=-0.8618). The viscosity of Pulp Canal Sealer EWT was the lowest and increased in the following order: AH Plus<Sealapex<Capseal (P<0.05). All of the tested root canal sealers showed characteristic time- and temperature-dependent changes in their rheological properties. The viscosities measured using the strain-controlled rheometer were more precise than the flowabilities measured using the simple press method, suggesting that the rheometer can accurately measure the rheological properties of root canal sealers.

Laser therapy in peri-implantitis treatment: literature review

Peri-implantitis is the most common reason for a late failure and can occur even after years of successful osseointegration. The role of microbial plaque accumulation in the development of peri-implantitis has been well documented. On the other hand, the ideal method of implant surface decontamination to re-establish the health of peri-implant tissue remains to be determined. Removal of bacterial deposits is essential in the treatment of peri-implant infections, and various therapeutic approaches have been described in the literature, including mechanical debridement, disinfection with chemotherapeutic agents, and laser therapy. Recently, there has been a plenitude of scientific data regarding the use of laser irradiation to achieve titanium surface decontamination. Thus, research is focusing on lasers' potential use in the treatment of peri-implantitis. The aim of this literature review is to analyze and evaluate the efficacy of laser therapy for the treatment of peri-implantitis.

Bacterial entombment by intratubular mineralization following orthograde mineral trioxide aggregate obturation: a scanning electron microscopy study / 国际口腔科学杂志·英文版

The time domain entombment of bacteria by intratubular mineralization following orthograde canal obturation with mineral trioxide aggregate (MTA) was studied by scanning electron microscopy (SEM). Single-rooted human premolars (n=60) were instrumented to an apical size #50/0.06 using ProFile and treated as follows: Group 1 (n=10) was filled with phosphate buffered saline (PBS); Group 2 (n=10) was incubated with Enterococcus faecalis for 3 weeks, and then filled with PBS; Group 3 (n=20) was obturated orthograde with a paste of OrthoMTA (BioMTA, Seoul, Korea) and PBS; and Group 4 (n=20) was incubated with E. faecalis for 3 weeks and then obturated with OrthoMTA-PBS paste. Following their treatments, the coronal openings were sealed with PBS-soaked cotton and intermediate restorative material (IRM), and the roots were then stored in PBS for 1, 2, 4, 8 or 16 weeks. After each incubation period, the roots were split and their dentin/MTA interfaces examined in both longitudinal and horizontal directions by SEM. There appeared to be an increase in intratubular mineralization over time in the OrthoMTA-filled roots (Groups 3 and 4). Furthermore, there was a gradual entombment of bacteria within the dentinal tubules in the E. faecalis inoculated MTA-filled roots (Group 4). Therefore, the orthograde obturation of root canals with OrthoMTA mixed with PBS may create a favorable environment for bacterial entombment by intratubular mineralization.

In-depth morphological study of mesiobuccal root canal systems in maxillary first molars: review

A common failure in endodontic treatment of the permanent maxillary first molars is likely to be caused by an inability to locate, clean, and obturate the second mesiobuccal (MB) canals. Because of the importance of knowledge on these additional canals, there have been numerous studies which investigated the maxillary first molar MB root canal morphology using in vivo and laboratory methods. In this article, the protocols, advantages and disadvantages of various methodologies for in-depth study of maxillary first molar MB root canal morphology were discussed. Furthermore, newly identified configuration types for the establishment of new classification system were suggested based on two image reformatting techniques of micro-computed tomography, which can be useful as a further 'Gold Standard' method for in-depth morphological study of complex root canal systems.

Antimicrobial effect of alexidine and chlorhexidine against Enterococcus faecalis infection / 国际口腔科学杂志·英文版

A previous study demonstrated that alexidine has greater affinity for the major virulence factors of bacteria than chlorhexidine. The aim of this study was to compare the antimicrobial activity of 1% alexidine with that of 2% chlorhexidine using Enterococcus faecalis-infected dentin blocks. Sixty bovine dentin blocks were prepared and randomly divided into six groups of 10 each. E. faecalis was inoculated on 60 dentin blocks using the Luppens apparatus for 24 h and then the dentin blocks were soaked in 2% chlorhexidine or 1% alexidine solutions for 5 and 10 min, respectively. Sterile saline was used as a control. The antimicrobial efficacy was assessed by counting the number of bacteria adhering to the dentin surface and observing the degradation of bacterial shape or membrane rupture under a scanning electron microscope. Significantly fewer bacteria were observed in the 2% chlorhexidine- or 1% alexidine-soaked groups than in the control group (P<0.05). However, there was no significant difference in the number of bacteria adhering to the dentinal surface between the two experimental groups or between the two soaking time groups (P>0.05). Ruptured or antiseptic-attached bacteria were more frequently observed in the 10-min-soaked chlorhexidine and alexidine groups than in the 5-min-soaked chlorhexidine and alexidine groups. In conclusion, 10-min soaking with 1% alexidine or 2% chlorhexidine can be effective against E. faecalis infection.

Comparison of gene expression profiles of human dental pulp cells treated with mineral trioxide aggregate and calcium hydroxide / 대한치과보존학회지

OBJECTIVES: This study investigated changes in gene expressions concerning of differentiation, proliferation, mineralization and inflammation using Human-8 expression bead arrays when white Mineral Trioxide Aggregate and calcium hydroxide-containing cement were applied in vitro to human dental pulp cells (HDPCs). MATERIALS AND METHODS: wMTA (white ProRoot MTA, Dentsply) and Dycal (Dentsply Caulk) in a Teflon tube (inner diameter 10 mm, height 1 mm) were applied to HDPCs. Empty tube-applied HDPCs were used as negative control. Total RNA was extracted at 3, 6, 9 and 24 hr after wMTA and Dycal application. The results of microarray were confirmed by reverse transcriptase polymerase chain reaction. RESULTS: Out of the 24,546 genes, 43 genes (e.g., BMP2, FOSB, THBS1, EDN1, IL11, COL10A1, TUFT1, HMOX1) were up-regulated greater than two-fold and 25 genes (e.g., SMAD6, TIMP2, DCN, SOCS2, CEBPD, KIAA1199) were down-regulated below 50% by wMTA. Two hundred thirty nine genes (e.g., BMP2, BMP6, SMAD6, IL11, FOS, VEGFA, PlGF, HMOX1, SOCS2, CEBPD, KIAA1199) were up-regulated greater than two-fold and 358 genes (e.g., EDN1, FGF) were down-regulated below 50% by Dycal. CONCLUSIONS: Both wMTA and Dycal induced changes in gene expressions related with differentiation and proliferation of pulp cells. wMTA induced changes in gene expressions related with mineralization, and Dycal induced those related with angiogenesis. The genes related with inflammation were more expressed by Dycal than by wMTA. It was confirmed that both wMTA and Dycal were able to induce gene expression changes concerned with the pulp repair in different ways.

Clinical assessment of whitening efficacy and safety of in-office tooth whitening system containing 15% hydrogen peroxide with or without light activation / 대한치과보존학회지

OBJECTIVES: This clinical study evaluated the effect of light activation on the whitening efficacy and safety of in-office bleaching system containing 15% hydrogen peroxide gel. MATERIALS AND METHODS: Thirty-three volunteers were randomly treated with (n = 17, experimental group) or without light activation (n = 16, control group), using Zoom2 white gel (15% H2O2, Discus Dental) for a total treatment time of 45 min. Visual and instrumental color measurements were obtained using Vitapan Classical shade guide and Shadepilot (DeguDent) at screening test, after bleaching, and 1 month and 3 month after bleaching. Data were analyzed using t-test, repeated measure ANOVA, and chi-squared test. RESULTS: Zoom2 white gel produced significant shade changes in both experimental and control group when pre-treatment shade was compared with that after bleaching. However, shade difference between two groups was not statistically significant (p > 0.05). Tooth shade relapse was not detected at 3 months after bleaching. The incidence of transient tooth sensitivity was 39.4%, with being no differences between two groups. CONCLUSIONS: The application of light activation with Zoom2 white gel system neither achieved additional whitening effects nor showed more detrimental influences.

Matrix metalloproteinase-8 and substance P levels in root canal exudates of nonvital teeth / 대한치과보존학회지

OBJECTIVES: The aim of this study was to investigate levels of matrix metalloproteinase-8 (MMP-8) and substance P (SP) in root canal exudates during root canal treatment (RCT) of nonvital, painful teeth. MATERIALS AND METHODS: Patients scheduled for nonsurgical RCT were prospectively selected; the study was performed after obtaining informed consent from the patients and was approved by the Institutional Review Board for Clinical Research of Gangnam Severance Hospital, Yonsei University (3-2008-0118). Canal exudates samples were collected using sterilized paper points from teeth scheduled for RCT across three different time periods. MMP-8 and SP levels were measured using enzyme-linked immunosorbent assay (ELISA). Data were analyzed using a mixed model analysis and the Pearson correlation analysis (p < 0.05). RESULTS: MMP-8 and SP levels in GCF were decreased during RCT (p < 0.0001), and they showed a weak positive correlation to each other (p < 0.05). Patients'subjective pain levels and the response from percussion test were significantly related to SP level. CONCLUSIONS: This study demonstrated that periradicular inflammation endodontic origin can elevate SP and MMP-8 levels in root canal exudates. Interestingly, SP level of canal exudates showed a possibility of being used as an indicator of pain due to periapical pathosis.

Microbial profile of asymptomatic and symptomatic teeth with primary endodontic infections by pyrosequencing / 대한치과보존학회지

OBJECTIVES: The purpose of this in vivo study was to investigate the microbial diversity in symptomatic and asymptomatic canals with primary endodontic infections by using GS FLX Titanium pyrosequencing. MATERIALS AND METHODS: Sequencing was performed on 6 teeth (symptomatic, n = 3; asymptomatic, n = 3) with primary endodontic infections. Amplicons from hypervariable region of the small-subunit ribosomal RNA gene were generated by polymerized chain reaction (PCR), and sequenced by means of the GS FLX Titanium pyrosequencing. RESULTS: On average, 10,639 and 45,455 16S rRNA sequences for asymptomatic and symptomatic teeth were obtained, respectively. Based on Ribosomal Database Project Classifier analysis, pyrosequencing identified the 141 bacterial genera in 13 phyla. The vast majority of sequences belonged to one of the seven phyla: Actinobacteria, Bacteroidetes, Firmicutes, Fusobacteria, Proteobacteria, Spirochetes, and Synergistetes. In genus level, Pyramidobacter, Streptococcus, and Leptotrichia constituted about 50% of microbial profile in asymptomatic teeth, whereas Neisseria, Propionibacterium, and Tessaracoccus were frequently found in symptomatic teeth (69%). Grouping the sequences in operational taxonomic units (3%) yielded 450 and 1,997 species level phylotypes in asymptomatic and symptomatic teeth, respectively. The total bacteria counts were significantly higher in symptomatic teeth than that of asymptomatic teeth (p < 0.05). CONCLUSIONS: GS FLX Titanium pyrosequencing could reveal a previously unidentified high bacterial diversity in primary endodontic infections.

A retrospective study of the intentionally replanted mandibular second molars with C-shaped root canal configurations / 대한치과보존학회지

OBJECTIVES: The purpose of this retrospective study was to evaluate the success rate of intentionally replanted mandibular second molar with C-shaped canal configurations and to access the impact of preoperative periapical lesion on the success of intentional replantation procedure. MATERIALS AND METHODS: This retrospective chart review study evaluated 52 intentionally replanted mandibular second molar teeth treated at Seoul National University Dental Hospital Department of Conservative Dentistry from January 2005 to December 2007. Seventeen teeth were lost for the follow-up, and another 6 teeth did not meet inclusion criteria of C-shaped root canal configurations. Healing outcome such as success, uncertain healing, and failure after follow-up was evaluated by clinical criteria and radiographs. RESULTS: The overall success rate was 72.4% for the 29 intentionally replanted C-shaped mandibular second molars. The success rate of replanted teeth with preoperative periapical lesions was similar to that of replanted teeth which have no periapical lesions. CONCLUSIONS: Therefore, root canal treatment failure on C-shaped mandibular second molar can be predictably treated by intentional replantation regardless of the presence of periapical lesion.

Analysis of para-chloroaniline after chemical interaction between alexidine and sodium hypochlorite using mass spectrometry: A preliminary study / 대한치과보존학회지

The purposes of this study were firstly to investigate the any formation of precipitate after interaction between ALX and NaOCL and secondarily to analyze the PCA formation by using time of flight secondary ion mass (TOF-SIM) spectrometry. Mass spectrometry analysis was performed for the mixture of 0.5% ALX and 5.25% NaOCl. As controls, 2.5% CHX with 5.25% NaOCl and 1% PCA solutions were used. Any formation of precipitates in 10 tested solutions was evaluated by naked eye. Results of mass spectrum showed that the typical peak of PCA was not detected in mixed solution of ALX and NaOCl, whereas CHX/NaOCl mixture showed the same peak that found in the PCA spectrum. Precipitate formation was only observed in CHX/NaOCL mixture. The present TOF-SIM spectrometry results indicated that ALX can be a useful root canal irrigant combined with NaOCl during canal instrumentation. Further study is necessary to confirm the antimicrobial effect of ALX against endodontic pathogen before its clinical application as an endodontic irrigant.

Apical prepration size in infected root canals / 대한치과보존학회지

The final preparation (MAF) size in infected root canals is still controversial. Nonetheless, recent studies demonstrated that larger apical preparation sizes produces a greater reduction in remaining bacteria and dentinal debris as compared to smaller apical preparation sizes. Therefore, clinicians should be practiced with treatment strategies guided by evidence-based information, especially in infected/failed root canals.

The relationship of radiographic lesion size and characteristics to diagnosis of periapical cysts and granulomas / 대한치과보존학회지

The purpose of this study was to find out the relationship of radiographic lesion size, gender, age of patients and radiographic character to the diagnosis of periapical cyst and granuloma. The data was collected from 187 periapical lesions of 167 patients who undergone apical surgery at Department of Conservative Dentistry, Seoul National University Dental Hospital from 2003 to 2005. The lesion were surgically removed and send for biopsy to the Oral Pathology Laboratory. From the initial radiograph, lesion size was calculated using PiViewSTAR(R) (INFINITT, Korea) program. The obtained data were statistically evaluated using SPSS (p 0.05). 4. There was a significant correlation between the non-demarcation of the lesion and the incidence of periapical granuloma (p < 0.01).

Gene expression profiling in human dental pulp cells treated with mineral trioxide aggregate / 대한치과보존학회지

This study investigated the changes in gene expression when mineral trioxide aggregate (MTA) was applied in vitro to human dental pulp cells (HDPCs). MTA in a teflon tube (diameter 10 mm, height 2 mm) was applied to HDPCs. Empty tube-applied HDPCs were used as negative control. For microarray analysis, total RNA was extracted at 6, 24, and 72 hrs after MTA application. The results were confirmed selectively by performing reverse transcriptase polymerase chain reaction for genes that showed changes of more than two-fold or less than half. Of the 24,546 genes, 109 genes were up-regulated greater than two-fold (e.g., FOSB, THBS1, BHLHB2, EDN1, IL11, FN1, COL10A1, and TUFT1) and 69 genes were down-regulated below 50% (e.g., SMAD6 and DCN). These results suggest that MTA, rather than being a bio-inert material, may have potential to affect the proliferation and differentiation of pulp cells in various ways.

Effect of cross-sectional area of 6 nickel-titanium rotary instruments on the fatigue fracture under cyclic flexural stress: A fractographic analysis / 대한치과보존학회지

This study aimed to assess the influence of different cross-sectional area on the cyclic fatigue fracture of Ni-Ti rotary files using a fatigue tester incorporating cyclical axial movement. Six brands of Ni-Ti rotary files (ISO 30 size with .04 taper) of 10 each were tested: Alpha system (KOMET), HeroShaper (MicroMega), K3 (SybronEndo), Mtwo (VDW), NRT (Mani), and ProFile (Dentsply). A fatigue-tester (Denbotix) was designed to allow cyclic tension and compressive stress on the tip of the instrument. Each file was mounted on a torque controlled motor (Aseptico) using a 1:20 reduction contra-angle and was rotated at 300 rpm with a continuous, 6 mm axial oscillating motion inside an artificial steel canal. The canal had a 60degrees angle and a 5 mm radius of curvature. Instrument fracture was visually detected and the time until fracture was recorded by a digital stop watch. The data were analyzed statistically. Fractographic analysis of all fractured surfaces was performed to determine the fracture modes using a scanning electron microscope. Cross-sectional area at 3 mm from the tip of 3 unused Ni-Ti instruments for each group was calculated using Image-Pro Plus (Imagej 1.34n, NIH). Results showed that NRT and ProFile had significantly longer time to fracture compared to the other groups (p < .05). The cross-sectional area was not significantly associated with fatigue resistance. Fractographycally, all fractured surfaces demonstrated a combination of ductile and brittle fracture. In conclusion, there was no significant relationship between fatigue resistance and the cross-sectional area of Ni-Ti instruments under experimental conditions.