Gene-expression analysis of cementoblasts and osteoblasts.

BACKGROUND AND OBJECTIVE: Cementum and bone are similar mineralized tissues, but cementum accumulates much more slowly than bone, does not have vasculature or innervation and does not undergo remodeling. Despite these differences, there are no well-established markers to distinguish cementoblasts from other mature mineralizing cells such as osteoblasts and odontoblasts. The purpose of this study was to assess differences in gene expression between cementoblasts and osteoblasts using gene profiling of cell populations isolated directly from osteocalcin-green fluorescent protein (OC-GFP) transgenic mice. MATERIAL AND METHODS: OC-GFP reporter mice were used as they show labeling of cementoblasts, osteoblasts and odontoblasts, but not of periodontal ligament fibroblasts, within the periodontium. We sorted cells digested from the molar root surface to isolate OC-GFP(+) cementoblasts. Osteoblasts were isolated from calvarial digests. Microarray analysis was performed, and selected results were confirmed by real-time PCR and immunostaining or in situ hybridization. RESULTS: Microarray analysis identified 95 genes that were expressed at least two-fold higher in cementoblasts than in osteoblasts. Our analysis indicated that the Wnt signaling pathway was differentially regulated, as were genes related to skeletal development. Real-time PCR confirmed that expression of the Wnt inhibitors Wnt inhibitory factor 1 (Wif1) and secreted frizzled-related protein 1 (Sfrp1) was elevated in cementoblasts compared with osteoblasts, and Wif1 expression was localized to the apical root region. In addition, the transcription factor BARX homeobox 1 (Barx1) was expressed at higher levels in cementoblasts, and immunohistochemistry indicated that BARX1 was expressed in apical cementoblasts and cementocytes, but not in osteoblasts or odontoblasts. CONCLUSION: The OC-GFP mouse provides a good model for selectively isolating cementoblasts, and allowed for identification of differentially expressed genes between cementoblasts and osteoblasts.

Three-dimensional modelling and concurrent measurements of root anatomy in mandibular first molar mesial roots using micro-computed tomography.

AIM: To obtain concurrent radicular measurements in the mesiobuccal (MB) and mesiolingual (ML) canals of mandibular first molars using scanned data of micro-computed tomography (µCT) with novel software. METHODOLOGY: The scanned data from 37 mandibular first molar mesial roots were reconstructed and analysed with custom-developed software (Kappa2). For each canal, three-dimensional (3D) surface models were re-sliced at 0.1-mm intervals perpendicular to the central axis. Dentine thicknesses, canal widths and 3D curvatures were measured automatically on each slice. Measurements were analysed statistically with anova for differences at each direction and at different levels of both canals. RESULTS: Lateral dentine thicknesses were significantly higher than mesial and distal thicknesses, at all the levels of both canals (P < 0.001). Mesial thicknesses were significantly higher than distal thicknesses in the coronal third of both canals (P < 0.001). Thinnest dentine thicknesses were mainly located on the disto-inside of both canals. Narrowest canal widths were 0.24 ± 0.10 and 0.22 ± 0.09 mm in MB and ML canals, respectively. Canal curvatures were greatest in the apical third of both canals (P < 0.001), and they were greater in the MB canals than in the ML canals (P < 0.05). CONCLUSIONS: Micro-computed tomography with novel software provided valuable anatomical information for optimizing instrumentation and minimizing mishaps in nonsurgical root canal treatment.

Autotransplantation of mesiodens for missing maxillary lateral incisor with cone-beam CT-fabricated model and orthodontics.

AIM: Autotransplantation is a viable treatment option for a missing tooth when there is a suitable donor, especially in adolescents with remaining facial growth. This report presents the aesthetic restoration of a missing maxillary lateral incisor through orthodontic treatment and autotransplantation of a mesiodens using a CBCT-fabricated rapid-prototyping model. SUMMARY: A 14-year-old male patient with a congenitally missing maxillary lateral incisor was referred from the Department of Orthodontics. The teeth were moved orthodontically to regain space for the missing lateral incisor and to close the space of the mesiodens after transplantation. A replica of the donor tooth was fabricated from a cone-beam computed tomography scan through a rapid-prototyping machine before autotransplantation surgery. The model was used to create a socket for the graft tooth, thereby shortening the extra-oral time and minimizing the damage to the root surface. After transplantation and orthodontic tooth movement, the mesiodens was finally restored with an aesthetic laminate restoration. Over 3 years, the aesthetics remained excellent, and the transplant functioned normally without any signs or symptoms of root resorption. KEY LEARNING POINT: Missing anterior teeth may be replaced through a combination of orthodontics, autotransplantation with a rapid-prototyping model and prosthodontic restoration, in growing patients.

Trace metal contents of three tricalcium silicate materials: MTA Angelus, Micro Mega MTA and Bioaggregate.

AIM: To investigate the levels of nine metals [aluminium (Al), antimony (Sb), arsenic (As), beryllium (Be), cadmium (Cd), chromium (Cr), iron (Fe), lead (Pb) and molybdenum (Mo)] in MTA Angelus, Micro Mega MTA and Bioaggregate using inductively coupled plasma-optical emission spectrometry (ICP-OES). METHODOLOGY: Each material (0.2 g) was digested using a mixture of hydrochloric and nitric acids and then filtered. The levels of nine metals in the resulting filtrates were measured by ICP-OES. The results were statistically analysed using one-way anova and the Bonferroni test. RESULTS: MTA Angelus contained more aluminium, beryllium and chromium than Micro Mega MTA (P < 0.05), whilst their levels of arsenic, cadmium and iron were similar. Antimony, lead and molybdenum were not detected in any of the three tested cements. Bioaggregate contained trace amounts of aluminium. CONCLUSIONS: MTA Angelus and Micro Mega MTA contained small amounts of seven tested metal oxides. Bioaggregate only contained trace amounts of aluminium.

Antimicrobial efficacy of a human ß-defensin-3 peptide using an Enterococcus faecalis dentine infection model.

AIM: To assess the antibacterial efficacy of a human ß-defensin-3 (HBD3) peptide against Enterococcus faecalis biofilms. METHODOLOGY: Standardized human dentine blocks were infected with E. faecalis ATCC 29212 for 3 weeks. Aqueous calcium hydroxide paste (n = 12, CH), a 2% chlorhexidine gel (n = 12, CHX), an HBD3 peptide gel (n = 12) and saline (n = 12) were tested as experimental groups. A mismatched peptide gel group (n = 12, MP) and sterilized but noninoculated block group (n = 12) were included as controls. After 1 week of medication, the dentinal samples at the depth of 200 and 400 µm were collected from medicated canal lumens. Bacterial growth was assessed by spectrophotometric analysis of optical density (OD) after 72 h of incubation. Statistical analysis was performed with repeated-measures anova and Tukey's post hoc test. RESULTS: The HBD3 group was associated with significantly lower OD values (P < 0.05) than the CH or CHX groups at both depths. The CH group did not differ significantly from MP or Saline group at either depth (P > 0.05). There was no significant difference (P > 0.05) in the OD values of the inner (200 µm) and outer (400 µm) dentinal samples for any group. CONCLUSIONS: The HBD3 peptide inhibited the growth of E. faecalis biofilms in infected dentine blocks.

A biometric study of C-shaped root canal systems in mandibular second molars using cone-beam computed tomography.

AIM: To investigate the configuration of C-shaped canals in mandibular second molars, canal wall thickness and the orientation of the thinnest area at 1-mm intervals from the canal orifice to the apex by using cone-beam computed tomographic (CBCT) images. METHODOLOGY: Three-dimensional CBCT images of 92 Korean mandibular second molars having C-shaped root canals were analysed to determine their configuration using a modification of Melton's classification, as well as the thinnest walls and their location. Associations between configuration type and distance from the canal orifice to the apex, as well as associations between the directional orientation of the thinnest root wall and distance from the canal orifice to the apex, were assessed by Fisher's exact test. Because serial measurements of minimum wall thicknesses were correlated with individual teeth, a mixed-effects analysis was applied. RESULTS: The most common configuration types were Melton's type I in the coronal region and Melton's type III in the apical region. Mean thicknesses of the thinnest root canal walls were 1.39 ± 0.38, 0.85 ± 0.25 and 0.77 ± 0.20 mm in the coronal, middle and apical regions, respectively. The thicker the root canal walls at the orifice region, the greater the decrease in thickness towards the apical region (P < 0.05), with the linguo-central root area being the thinnest. The pattern of decreasing thickness from the orifice to the apex formed a nonlinear cubic curve. CONCLUSIONS: The most prevalent configuration types were Melton's type I (coronal region) and type III (apical region). The linguo-central root area was the thinnest in C-shaped root canals of Korean mandibular second molars. These anatomical variations should be considered during surgical or nonsurgical endodontic procedures.

Maxillary molar root and canal morphology of Neolithic and modern Chinese.

OBJECTIVE: This study aimed to characterize Neolithic human maxillary molars from archeological remains at the Jiaojia site, Shandong, China, and compare their ultrastructural features with sex and age-matched modern locals. DESIGN: Maxillary first (n = 86) and second (n = 80) molars in 5000-year-old individuals (n = 50) from the Jiaojia site were scanned by cone-beam computed tomography (CBCT). Sex and age-matched control groups were assigned from oral surgical patients at Shandong University. Images were analyzed for crown size, root length, root morphology, canal inter-orifice distances, mesiobuccal canal morphology, and second mesiobuccal (MB2) canal prevalence and location. Neolithic and modern values were compared statistically using Chi-squared and Mann-Whitney test at p < .05. RESULTS: Crown and root size were smaller, and canal inter-orifice distances were shorter in Neolithic maxillary molars than their modern counterparts. For mesiobuccal roots, Weine's Type I single canals were the most prevalent in Neolithic and modern first and second molars. MB2 canal prevalence were not significantly different (p > .05) in Neolithic (53.3%) or modern (60.5%) first molars, and Neolithic (11.3%) or modern (21.3%) second molars. But, MB2 prevalence was significantly higher for modern than ancient male first (p = .032) and second (p = .005) molars. Additionally, MB2 were located more mesially and closer to MB1 in Neolithic than modern molars. CONCLUSIONS: Maxillary molar root and canal morphology of ancient 5000-year-old remains at the Jiaojia site resemble that of local patients. A trend towards larger tooth size, and more dispersed MB2 canals over this short evolutionary period warrants additional investigation.

Cyclic fatigue resistance, torsional resistance, and metallurgical characteristics of V taper 2 and V taper 2H rotary NiTi files.

The aim of this study was to compare the cyclic fatigue resistance, torsional resistance, and metallurgical characteristics of conventional NiTi wire (V taper 2, V2) and CM wire (V taper 2H, V2H)-based files. Cyclic fatigue and torsional resistance of V2 and V2H were investigated by measuring the number of cycles to fracture, maximum torque at fracture, and maximum angle at fracture. The typical patterns of fatigue and torsional fractures were investigated using a scanning electron microscope (SEM). The metallurgical characteristics were investigated by differential scanning calorimetry (DSC) from -100 °C to 100 °C. The austenite finishing temperature (Af) of each instrument was also measured. The microstructures of the instruments were investigated by a transmission electron microscope (TEM) along with selected area diffraction pattern analysis. The results were statistically analyzed by Mann-Whitney U-test (p = 0.05). V2H showed significantly higher cyclic fatigue resistance and torsional resistance than V2. SEM images of the fractured surfaces showed typical patterns of fatigue and torsional fracture. The DSC analysis of V2 showed one small peak in both the heating and cooling curves. The Af of V2 was -0.32 °C. V2H showed two remarkable peaks in the heating curve and one remarkable peak in the cooling curve. The Af of V2H was 33.25 °C. The TEM analysis showed that both V2 and V2H are mainly composed of austenite. In conclusion, V2H showed higher cyclic fatigue resistance and torsional resistance than V2. The superior properties of V2H could be attributed to the annealing effect and possibly the martensite phase. SCANNING 38:564-570, 2016. © 2016 Wiley Periodicals, Inc.

Pyrosequencing Analysis of Subgingival Microbiota in Distinct Periodontal Conditions.

Subgingival microorganisms are potentially associated with periodontal diseases. However, changes in the subgingival microbiota during the progress of periodontal diseases are poorly understood. In this study, we analyzed bacterial communities in the subgingival paper point samples from 32 Korean individuals with no sign of disease, gingivitis, or periodontitis using 454 FLX Titanium pyrosequencing. A total of 256,113 reads representing 26 phyla, 433 genera, and 1,016 species were detected. Bacteroidetes, Fusobacteria, Synergistetes, and Spirochaetes were the abundant phyla in periodontitis subjects, whereas Firmicutes and Proteobacteria were identified as the dominant phyla in the gingivitis and healthy subjects, respectively. Although high levels of Porphyromonas, Fusobacterium, Fretibacterium, Rothia, Filifactor, and Treponema genera were observed in the periodontitis subjects, Streptococcus, Capnocytophaga, Leptotrichia, and Haemophilus genera were found at high frequency in the gingivitis subjects. Species including Porphyromonas gingivalis, Fusobacterium nucleatum, and Fretibacterium fastidiosum were significantly increased in periodontitis subjects. On the other hand, Streptococcus pseudopneumoniae, Haemophilus parainfluenzae, and Leptotrichia hongkongensis were preferentially observed in the gingivitis subjects. Intriguingly, the halophile Halomonas hamiltonii was revealed as a predominant species in the healthy subjects. Based on Fast UniFrac analysis, distinctive bacterial clusters were classified for the healthy, gingivitis, and periodontitis state. The current findings might be useful for understanding the pathogenesis, diagnosis, and treatment of periodontal diseases.

Molecular epidemiology and association of putative pathogens in root canal infection.

The purpose of this study was to investigate the epidemiology of putative pathogens in root canals with apical periodontitis and to determine the associations among the putative pathogens. Eighteen symptomatic and 20 asymptomatic teeth from 36 subjects were studied. This research was performed with polymerase chain reaction and hybridization using rRNA-based oligonucleotide probes. The most frequently found species was Fusobacterium sp. (68.4%), followed by Peptostreptococcus micros (44.7%) and Porphyromonas gingivalis (26.3%). Sixteen teeth (42.1%) contained one or more species of the selected black-pigmented bacteria. Bacteroides forsythus and Treponema sp. were detected in 8 teeth and 6 teeth, respectively. Among the analyzed bacteria, significant relationships were shown in the combination of B. forsythus/P. gingivalis and Treponema sp./P. gingivalis. There was no significant association between any bacteria and any symptoms.

Shaping ability of three ProFile rotary instrumentation techniques in simulated resin root canals.

The aim of this study was to compare the shaping ability of three ProFile rotary instrumentation techniques and a conventional step-back method in simulated root canals. Prevalence of canal aberrations, change in working length, and preparation time were measured. A total of 48 composite images were made from pre- and postcanal scanned images using Corel Photopaint 8.0 and then the amount of coronal substance the instruments removed was also calculated two-dimensionally on digitized images with the Brain C software to compare the enlarging efficiency. There were no significant differences between the three rotary groups in preparation time, change in working length, and the incidence of aberrations (p > 0.05). The amount of coronal substance the instruments removed in the ProFile .04 taper group was significantly smaller than the other three groups (p < 0.05).

Identification of oral spirochetes at the species level and their association with other bacteria in endodontic infections.

OBJECTIVES: Recent molecular approaches have revealed that fastidious organisms such as Bacteroides forsythus and oral treponemes were frequently found in root canals with apical periodontitis. The purpose of this study was to identify the isolates of oral spirochetes at the species level in endodontic infections and to determine their association with B forsythus and Porphyromonas gingivalis. STUDY DESIGN: Seventy-nine teeth with apical periodontitis were selected for this study. After sampling from the root canals aseptically, polymerase chain reaction amplification for the 16S rRNA gene was performed with eubacterial universal primers. Subsequently, dot-blot hybridization was performed with 8 species-specific oligonucleotide probes. The microbial associations were analyzed by using the odds ratio. RESULTS: The most frequently found species was P gingivalis (27.4%), followed by Treponema maltophilum (26%), B forsythus (16.4%), and Treponema socranskii (2.7%). Other treponemes, including Treponema denticola, were not detected in our samples. Significant microbial associations were identified between T maltophilum, B forsythus, and P gingivalis by performing analysis with the odds ratio. CONCLUSION: Our results indicate that T maltophilum should be included in etiologic studies of endodontic diseases.

Alpha-amylase is a human salivary protein with affinity to lipopolysaccharide of Aggregatibacter actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans lipopolysaccharide (Aa.LPS) is a major virulence factor associated with aggressive periodontitis. Although the recognition of Aa.LPS is potentially initiated by salivary proteins in the oral cavity, Aa.LPS-binding proteins (Aa.LPS-BPs) in saliva are poorly characterized. The purpose of this study was to capture and identify Aa.LPS-BPs in human saliva using a LTQ-Orbitrap hybrid Fourier transform mass spectrometry. Aa.LPS conjugated onto N-hydroxysuccinimidyl-Sepharose(®) 4 Fast Flow beads (Aa.LPS-beads) activated Toll-like receptor 4 and produced nitric oxide and Interferon gamma-inducible protein-10, implying that the conjugation process did not alter the biological properties of Aa.LPS. Aa.LPS-BPs were subsequently isolated from the nine human saliva samples from healthy individuals with the Aa.LPS-beads followed by identification with the mass spectrometry. Aa.LPS-BPs include α-amylase, serum albumin, cystatin, lysozyme C, submaxillary gland androgen-regulated protein 3B, immunoglobulin subunits, polymeric immunoglobulin receptor, deleted in malignant brain tumors 1, prolactin-inducible protein, lipocalin-1, and basic salivary proline-rich protein 2. Specific binding was validated using a pull-down assay with α-amylase which was captured at the highest frequency. Alpha-amylase demonstrated to interfere with the adherence and biofilm formation of A. actinomycetemcomitans. Even heat-inactivated α-amylase showed the interference to the same extent. Conclusively, we identified unique Aa.LPS-BPs that provide useful information to understand bacterial pathogenesis and host innate immunity in the oral cavity.

Minimum-intensity projection for in-depth morphology study of mesiobuccal root.

OBJECTIVE: This micro-computed tomography (MCT) study investigated the utility of thin-slab minimum-intensity projection (TS-MinIP) technique as an adjunct to 3-dimensional (3D) modeling for in-depth morphology study. STUDY DESIGN: One hundred one extracted maxillary first molars were scanned for microtomographic analysis (SkyScan). Two-dimensional TS-MinIP and 3D images of mesiobuccal (MB) roots were produced and analyzed to record the number and configurations of the canals, the incidence and location of accessory canals, loop, and intercanal connections, and number of foramina. RESULTS: Multiple-canal MB roots were present in 76.2%, and all of the roots had intercanal communications. Weine type III configuration was the most common in the multiple-canal roots. Accessory canals were found in 78.2% of the roots. Configurations that were nonclassifiable were found in 10.9% of the MB roots. CONCLUSIONS: MB root canal anatomy was complex, and MinIP may serve as an adjunct to 3D modeling for in-depth morphology study.

Clinical application of computer-aided rapid prototyping for tooth transplantation.

The maintenance of healthy periodontal ligament cells in the donor tooth is one of the most important factors for successful tooth transplantation. This is achieved by minimizing the extraoral time during the surgical procedure. If a duplicate form of donor tooth could be obtained before extraction, it would be possible to precontour the recipient alveolar bone compatible with the donor tooth, and thereby reduce the extra-oral time of the donor tooth. We obtained a three-dimensional image with the real dimensions of the donor tooth from a CT Highspeed Advantage, allowing a life-sized resin model of the tooth to be fabricated. From 22 clinical cases, we achieved an average total transplantation time of 7.7 min. The average distance between the transplanted root surface and the alveolar bone from 12 available cases was 0.87 mm at the mesial cervix, 0.91 mm at the mesial apex, 0.98 mm at the distal cervix and 1.16 mm at the distal apex on the postoperative radiographs. Clinical data indicate that computer-aided rapid prototyping may be of value in minimizing the extra-oral time and possible injury to transplanted tooth during the process of autotransplantation.

Molecular characterization of the presence of Eubacterium spp and Streptococcus spp in endodontic infections.

Eubacterium spp. and Streptococcus spp. are virulent, commonly identified microorganisms in endodontic infections. The purpose of this study was to use molecular methods to identify these organisms in 22 infected root canals that include eight cases with preoperative clinical symptoms and five cases with a history of diabetes mellitus. The presence of Streptococcus spp. and Eubacterium spp. was examined using two sets of PCR primers specific with multiple species within the respective genera. Positive specimens had their PCR products sequenced and phylogenetically analyzed to identify the specific species. Sixteen specimens (73%) contained Eubacterium spp. and nine (41%) were positive for Streptococcus spp. Eubacterium infirmum was the most prevalent Eubacterium sp. This organism was significantly associated with a history of diabetes (OR = 9.6; P = 0.04). Streptococcus anginosus was the most common Streptococcus sp., but neither it nor any of the other streptococci were significantly associated with the clinical parameters evaluated.