Caiman periodontium as an intermediate between basal vertebrate ankylosis-type attachment and mammalian "true" periodontium

The teeth of many fish, amphibia, and reptiles are attached to the alveolar bone via ankylosis. In contrast, mammalian periodontia are characterized by a gomphosis, an attachment of the tooth root in the alveolar bone socket via periodontal ligament fibers. Among the reptiles, the crocodilians are the only group featuring a gomphosis-type connection between tooth root and alveolar bone, while in other reptiles tooth-root and jawbone are connected via ankylosis. The purpose of the present study was to compare several key features of the crocodilian periodontium with those of the mammalian and noncrocodilian reptile periodontium. As experimental models for our study we chose the periodontium of newborn geckos (Hemidacylus turcicus), juvenile caimans (Caiman crocodilus crocodilus), and 10-day-postnatal Swiss-Webster mice (Mus musculus) as representative models for noncrocodilian reptiles, crocodilian reptiles, and mammals. The caiman periodontium emerged as an intermediary between the mineral-free mouse ligament and the mineralized gecko ankylosis-type attachment. Caiman ligament fibers were less organized than mouse ligament fibers but featured distinct fasciae surrounding ligament fiber bundles. Caiman Hertwig's epithelial root sheath (HERS) was similarly perforated as mouse HERS and distinctly different from the continuous gecko HERS. Both caiman and mouse HERS covered the entire tooth root length, while in the gecko HERS was limited to the coronal portion of the root, allowing for cementoid-mediated ankylosis at the apical tip of the root. We interpret our data to indicate distinct differences in mineral distribution, periodontal ligament fiber organization, and HERS distribution between noncrocodilian reptiles, crocodilian reptiles, and mammals. Mineral deposits in the caiman ligament may reflect an evolutionary position of the caiman periodontium between ankylosis and gomphosis.     

Periodontal research contributions to basic sciences: From cell communication and host-parasite interactions to inflammation and bone biology

The periodontium comprises all structures surrounding the teeth, including gingiva, root cementum, periodontal ligament and alveolar bone. Those tissues aim to protect and support the teeth and are challenged by a residing microbiota that leads to subclinical inflammation even in physiological conditions. Periodontitis, a prevalent multicausal inflammatory and destructive disease, develops as a result from complex host-parasite interactions. This unique physiologic and pathologic scenario enables the development of research methods which allows conclusions beyond the simple understanding of periodontal homeostasis. The aim of this viewpoint was to explore potential contributions of periodontal research to a wide array of basic science specialties, such as cell and molecular biology, microbiology, immunology, endocrinology, rheumatology, among others.     

Regulatory mechanisms of periodontal regeneration

The periodontal ligament, located between the cementum and the alveolar bone, has a width ranging from 0.15 to 0.38 mm. Regeneration and homeostasis of the periodontal ligament are highly significant functions in relation to periodontal therapy, tooth transplantation or replantation, and orthodontic tooth movement. The purpose of this review is to discuss the regulatory mechanisms of regenerative and homeostatic functions in the periodontal ligament based on currently published studies and also on our own experimental data. We consider the capability of the ligament tissue to promote or to suppress calcification in connection with bone and cementum formation and the maintenance of the periodontal ligament space. Also discussed are the involvement of the periodontal ligament tissue in the regenerative ability, cell proliferation, growth and differentiation factors, extracellular matrix proteins, homeostatic phenomena, function of Malassez epithelial rests, tooth movement, or occlusal loading. Regulatory mechanisms for regeneration and homeostasis of the periodontal ligament are hypothetically proposed.     

Resin luting materials: Tissue response in dog's teeth

The aim of this study was to evaluate radiographically and histologically the pulpal and periapical response to self‐adhesive (Rely X? Unicem) and self‐etching and self‐curing (Multilink^®) resin‐based luting materials in deep cavities in dogs' teeth. Deep class V cavities (0.5‐mm–thick dentin) were prepared in 60 canine premolars and the following materials were applied on cavity floor: Groups I/V—RelyX? Unicem; Groups II/VI—Multilink^®; Groups III/VII—zinc phosphate cement (control) and; Groups IV/VIII—gutta‐percha (control). Cavities were restored with silver amalgam. Animals were euthanized after 10 days (groups I–IV) and 90 days (groups V–VIII). Tooth/bone blocks were radiographed and processed for histopathological evaluation of pulp and periapical tissue response to the materials. All materials presented similar histopathological features and radiographic findings at both periods. The pulp tissue was intact. The apical and periapical regions and periodontal ligament thickness were normal. No inflammatory cells, resorption of mineralized tissue (dentin, cementum, and alveolar bone) or bacteria were observed. The lamina dura was intact and no areas of periapical bone rarefaction or internal/external root resorption were observed radiographically. It can be concluded that Rely X? Unicem and Multilink^® caused no adverse tissue reactions and may be indicated for cementation of indirect restorations in deep dentin cavities without pulp exposure. Microsc. Res. Tech. 78:1098–1103, 2015. © 2015 Wiley Periodicals, Inc.     

How effective is ‘high-speed’ autoradiography?

A number of methods have recently been described for enhancing silver grain production in light microscope autoradiography by impregnating the emulsion with a 2,5-diphenyloxazole-based fluor system. After extensive testing of these methods it was found that none gave a significant increase in silver grain count. In the course of this work a number of observations were made which seem to account at least partially for these failures, and which question the feasibility of this type of fluorography.

• 1 Tetrahymena nuclei, pulse labelled with ³H-thymidine, contain sufficient radioactivity to produce useful autoradiographs after 2 h exposure without fluorographic enhancement.
• 2 Although fluorographic autoradiography of this type requires that the fluor penetrate the photographic emulsion, direct evidence was obtained that such penetration does not occur. Thus it is unlikely that true fluorographic enhancement occurs with methods based on 2,5-diphenyloxazole impregnation of the emulsion. (This is in contrast to the widely used gel fluorography techniques in which it is the gel rather than the photographic emulsion which is impregnated with fluor.)
• 3 In DNA fibre autoradiography, it is possible to obtain artefactual arrays of silver grains which closely resemble those expected of replicating DNA. This phenomenon alone, however, does not explain a recent report of successful DNA fibre autoradiography after an exposure of only 3 days. Calculations based on the maximum dpm per length of labelled DNA raise further questions concerning this result.

     

Time-dependent mechanical behaviour of the periodontal ligament

The process of tooth displacement in response to orthodontic forces is thought to be induced by the stresses and strains in the periodontium. The mechanical force on the tooth is transmitted to the alveolar bone through a layer of soft connective tissue, the periodontal ligament. Stress and/or strain distribution in this layer must be derived from mathematical models, such as the finite element method, because it cannot be measured directly in a non-destructive way. The material behaviour of the constituent tissues is required as an input for such a model. The purpose of this study was to determine the time-dependent mechanical behaviour of the periodontal ligament due to orthodontic loading of a tooth. Therefore, in vivo experiments were performed on beagle dogs. The experimental configuration was simulated in a finite element model to estimate the poroelastic material properties for the periodontal ligament. The experiments showed a two-step response: an instantaneous displacement of 14.10 +/- 3.21 microns within 4 s and a more gradual (creep) displacement reaching a maximum of 60.00 +/- 9.92 microns after 5 h. This response fitted excellently in the finite element model when 21 per cent of the ligament volume was assigned a permeability of 1.0 x 10(-14) m4/N s, the remaining 97 per cent was assigned a permeability of 2.5 x 10(-17) m4/N s. A tissue elastic modulus of 0.015 +/- 0.001 MPa was estimated. Our results indicate that fluid compartments within the periodontal ligament play an important role in the transmission and damping of forces acting on teeth.     

Lymphatic networks in the periodontal tissue and dental pulp as revealed by histochemical study.

The structural organization and fine distribution of the lymphatic networks in the periodontal tissues (gingiva, periodontal membrane, and alveolar process) and dental pulp of animals and humans were reviewed with special reference to histochemical examination by light and electron microscopy. The distinction between lymphatics and blood vessels was made on cryostat sections of undecalcified and calcified teeth treated with EDTA solution and whole mount preparations of periodontal membranes using 5'-nucleotidase (5'-Nase)-alkaline phosphatase (ALPase) double staining. This staining procedure allowed lymphatic vessels in the periodontal tissue and dental pulp to be differentiated from blood vessels. The specificity and localization of the enzyme reactions were confirmed by comparative histochemical studies of the same specimen with light microscopy and scanning or transmission electron microscopy. Well-developed 5'-Nase-positive lymphatic networks were observed on the tissue sections and whole mount preparations of the gingiva, periodontium, and dental pulp. More lymphatic vessels were seen in the root area of the periodontium than in the cervical area. In the dental pulp, lymphatic vessels were more numerous in the central part than in the peripheral odontoblastic layer. These distributions of the lymphatic capillary networks are discussed in relation to their ability to supply lymph to the teeth.     

Regeneration of periodontal Ruffini endings in adults and neonates

We reviewed the regeneration of periodontal Ruffini endings, primary mechanoreceptors in the periodontal ligament, following injury to the inferior alveolar nerve (IAN) in adult and neonatal rats. Morphologically, mature Ruffini endings are characterized by an extensive arborization of axonal terminals and association with specialized Schwann cells, called lamellar or terminal Schwann cells. Following injury to IAN in the adult, the periodontal Ruffini endings of the rat lower incisor ligament regenerate more rapidly than Ruffini endings in other tissues. During regeneration, terminal Schwann cells migrate into regions where they are never found under normal conditions. The development of periodontal Ruffini endings of the rat incisor is closely associated with the eruption of the teeth; the morphology and distribution of the terminal Schwann cells became almost identical to those in adults during postnatal days 15-18 (PN 15-18d) when the first molars appear in the oral cavity, while the axonal elements showed extensive ramification around PN 28d when the functional occlusion commences. When the IAN was injured in neonates, the regeneration of periodontal Ruffini endings was delayed compared with the adults. The migration of terminal Schwann cells is also observed following IAN injury, after which the distribution of terminal Schwann cells became almost identical to that of the adults, i.e., PN 14d. Since the interaction between axon and Schwann cell is important during regeneration and development, further studies are required to elucidate its molecular mechanism during the regeneration as well as the development of the periodontal Ruffini endings.     

Back-scattered electron imaging for leakage analysis of four retrofilling materials

This study aimed to evaluate, ex vivo, the nanoleakage in dentinal tubules, the linear infiltration of silver nitrate in the dentin wall/root-end filling material interface, and the presence of gaps in this interface in root-end cavities filled with 4 filling materials. Forty-eight disto-buccal root canals of maxillary molars were instrumented and filled. Retrograde cavities were prepared with ultrasonic points (apical 2 mm). The samples were divided into 2 control groups (n = 4) and 4 experimental groups (n = 10): Group I--white mineral trioxide aggregate (MTA); Group II--Super EBA; Group III--Portland cement; and Group IV--Sealer 26. After 1 week, the specimens were subjected to silver nitrate and prepared for SEM (backscattered electrons). In the apical-apical segment, an area with significantly higher leakage was observed for Super EBA, followed by Portland cement, MTA, and Sealer 26 (P = 0.0054). In the medium and cervical segments, all materials showed the same leakage behavior (P = 0.1815 and P = 0.1723, respectively). The linear infiltration at the dentin wall/root-end filling material interface was higher with Super EBA than the other groups. No differences in the percentage of gaps along the 3 mm of dentin wall/root-end filling material interface between the 4 materials were evident (P > 0.05). Nanoleakage occurred mainly in the apical segment of the samples, and Super EBA showed the highest values. The area and linear leakage were lower in the middle and coronal segments, regardless of the root-end filling material. No material perfectly sealed the root-end cavities, which allowed for the leakage occurrence.     

Scanning electron microscopic observation of microvasculature in periodontium.

In this study, vascular resin cast models in the periodontium of beagle dogs were prepared and three-dimensional observation of the relationship between the gingiva and periodontal ligament (PDL) vascular network was performed. After the perfusion of Ringer's solution and fixative, synthetic resin was injected from the inferior alveolar arteries. Soft tissue was digested by proteinase solution and specimens were examined under scanning electron microscope (SEM). The gingival vascular network (GVN) in the region facing the teeth consisted of sulcular and junctional epithelium. The vascular network of the sulcular epithelium (SE) had a renal glomerulus-like form and the junctional epithelium (JE) consisted of squamous mesh. The gingival sulcular fluid exudated from the vascular network directly beneath the JE, and leukocytes permeated from the vascular network beneath the epithelium. Thus, we considered that the GVN performs an important function in the protection against the inflammation. Periodontal ligament had a polygonal mesh vascular network that was anastomosed to the venous plexus of alveolar bone through Volkmann's canals (VC). When occlusal force was applied, the blood in the periodontal vessels flowed out through VC into the bone marrow, and when the force was removed, it flowed backward into the PDL. This blood transfer acted as an absorber against occlusal force. Our findings suggest that the blood vessels of the gingiva perform an important function in defending against inflammation, while the blood vessels of the PDL play a key role in absorbing occlusal force.     

Residues of calcium hydroxide-based intracanal medication associated with different vehicles: a scanning electron microscopy evaluation

This study evaluated the presence of residues after removal of calcium hydroxide [Ca(OH)(2) ] associated with different vehicles. Thirty single-rooted teeth were instrumented to a master apical file #25 using 2.5% NaOCl as main irrigant and 17% trisodium EDTA (ethylenediaminetetraacetic acid) as final agent irrigant. Then, the root canals were dressed with Ca(OH)(2) associated with silicone oil (Group 1), 2% chlorhexidine gluconate (Group 2), or propylene glycol (Group 3). After coronal sealing, all teeth were kept in a moist environment at room temperature. After 7 days, the teeth were reopened and medicaments were removed using 5 mL of saline solution and instrumentation with master apical file followed by new irrigation with 5 mL of 2.5% NaOCl. Subsequently, teeth were split longitudinally and assessed by scanning electron microscopy. The wall cleanliness of the cervical and apical thirds of the roots were evaluated and scored by three blinded examiners. Statistical analysis was performed using Kruskal-Wallis and Wilcoxon tests at 5% level of significance. All roots had residues of Ca(OH)(2) on the canal walls. All experimental groups had similar results (P > 0.05) regardless of the third evaluated. There was significant difference between the apical and cervical thirds only in Group 3 (P < 0.05). Association of different vehicles to Ca(OH)(2) does not influence the persistence of residues on the root canal walls.     

Natural frequency analysis of tooth stability under various simulated types and degrees of alveolar vertical bone loss

The aim of this study was to test natural teeth stability under various simulated types and degrees of alveolar vertical bone loss, as well as to assess the role that the surrounding bone played for maintaining tooth stability. A three-dimensional finite element model of the human maxillary central incisor with surrounding tissue, including periodontal ligament, enamel, dentin, pulp, and alveolar bone, was established. One side and multiple vertical bone loss were simulated by means of decreasing the surrounding bone level apically from the cemento-enamel junction in 1 mm steps incrementally downward for 10 mm. Natural frequency values of the incisor model with various types and degrees of bone loss were then calculated. The results showed that, with one-sided bone resorption, the model with labial bone loss had the lowest natural frequency decreasing rates (8.2 per cent). On the other hand, in cases of multiple bone loss, vertical bone resorption at the mesial and distal sides had more negative effects on tooth stability compared to vertical bone losses on facial and lingual sides. These findings suggest that the natural frequency method may be a useful, auxiliary clinical tool for diagnosis of vertical periodontal diseases.     

Silver staining of collagen and reticulin fibres and crebral capillaries by means of physical development

A method for demonstrating collagen and reticulin fibres and the network of brain capillaries has been worked out on the basis of a new silver staining principle. It may be applied to sections as thick as 200 μm from both frozen and embedded material. The principle is as follows: the 1,2-glycol groups of tissue elements, including connective tissues, are converted to aldehyde groups by periodic acid. The aldehyde groups form colloidal silver particles in an ammoniated silver nitrate solution of pH 13. At this pH value, however, it is only the connective tissue that can bind the colloidal silver which is being formed. After the silver ions have been washed out of the sections, the silver grains deposited in the connective tissue are finally enlarged to microscopic dimensions by physical development.     

A methodological basis for SEM autoradiography: biosynthesis and radioligand binding

A method is described for scanning electron microscope (SEM) autoradiography whereby preservation of high resolution cell surface details is retained together with degelatination of the emulsion without gross loss or redistribution of silver grains. This method should provide a convenient medium-sized marker for SEM (using secondary, backscattered electron and X-ray imaging) topographic studies of biosynthesized molecules, and of cell surface receptors and antigens, using indirect or direct labelling procedures with radio-labelled ligands.     

Clinical microscopic analysis of protaper retreatment system efficacy considering root canal thirds using three endodontic sealers

To evaluate the efficacy of ProTaper Universal rotary retreatment system and the influence of sealer type on the presence of filling debris in the reinstrumented canals viewed in an operative clinical microscope. Forty‐five palatal root canals of first molars were filled with gutta‐percha and one of the following sealers: G1, EndoFill; G2, AH Plus; G3, Sealapex. The canals were then reinstrumented with ProTaper Universal rotary system. Roots were longitudinally sectioned and examined under an operative clinical microscope (10×), and the amount of filling debris on canal walls was analyzed using the AutoCAD 2004 software. A single operator used a specific software tool to outline the canal area and the filling debris area in each third (cervical, middle, and apical), as well as the total canal area. Data were analyzed by Kruskal‐Wallis test and Tukey test at P < 0.05. Sealapex demonstrated significant differences in the average of filling debris area/canal among the 3 thirds. This group revealed that apical third showed more debris than the both cervical and middle third (P < 0.0001). Endofill presented significantly more filling debris than Sealapex in the cervical third (P < 0.05). In the middle (P = 0.12) and apical third (P = 0.10), there were no differences amongst groups. Debris was left in all canal thirds, regardless of the retreatment technique. The greatest differences between techniques and sealers were found in the cervical third. Microsc. Res. Tech. 75:1233–1236, 2012. © 2012 Wiley Periodicals, Inc.     

Quantitative evaluation of autoradiographs by X-ray spectroscopy.

Measurements of mass of developed silver in autoradiograms have been made using X-ray microanalysis. This procedure has been applied to a scanning electron microscopic study of protease-induced cell stimulation in 3H-thymidine-labelled bladder tissues in vitro. Differences in silver concentrations over individual nuclei were demonstrated by X-ray analysis and quantitative data was obtained showing an increased rate of 3H-thymidine incorporation in protease-treated cultures. The initial evidence indicates that X-ray microanalysis could provide a potentially useful quantitative procedure for autoradiography.     

Percentage of gutta-percha in mesial canals of mandibular molars obturated by lateral compaction or single cone techniques

The aim of this study was to compare the percentage of gutta‐percha (PGP) in mesial root canals of mandibular molars obturated with LC (Lateral Compaction) or SC (Single Cone) ProTaper Universal System techniques at different levels of the root. Mesial root canals of 20 human permanent molars with similar anatomical characteristics were instrumented using the ProTaper Universal rotary system technique until the F2 instrument, with 20 canals filled by SC ProTaper Universal technique and 20 canals by the LC technique. The mesial roots were sectioned transversely to 3, 5, and 7 mm from the root apex. Digital images of specimens were obtained at MIC‐D digital microscope in increases of 30 to 35X. The gutta‐percha area was measured using ImageTool software. Data were analyzed using two‐way ANOVA and Bonferroni test (α = 0.05). The SC technique provided greater PGP than the LC technique in the apical third (3 mm) (P < 0.001). In the other thirds (5 and 7 mm) there was no statistical difference between the two techniques regarding the PGP (P > 0.05). There was no statistically significant difference between thirds of the root canal for both techniques (P > 0.05). It was concluded that SC technique provided greater PGP than the LC technique in the apical third of mesial root canals of mandibular molars. There was no difference between the two techniques regarding the PGP in the cervical and middle thirds. Microsc. Res. Tech. 75:1229–1232, 2012. © 2012 Wiley Periodicals, Inc.     

A rapid method for identification of S-phase cells in transmission electron microscopy of monolayer cultures

A rapid technique has been developed for identifying cells in S-phase in transmission electron microscopy of monolayer cultures. Autoradiography is performed on the embedded monolayer which is then re-embedded. The cells and the adjacent layer of developed emulsion are sectioned together, and when subsequently viewed in the electron microscope the cells in S-phase appear with a zone of silver grains in the emulsion which is opposite their nuclei. They are thus readily identified and can be selected for detailed study. The advantages of this technique over conventional TEM autoradiography are the very much shorter exposure times and a reduced workload when preparing serial sections.     

The effect of a root‐dentin pretreatment technique combining PIPS with MTAD aiming to improve the bond strength of glass fiber post

The aim of this study was to evaluate the combined effect of MTAD and photon‐induced photoacoustic streaming (PIPS) technique on the smear layer removal and the bond strength of glass fiber post. Fifty‐five human mandibular premolars were chosen. After root canal therapy and post space preparation, the teeth were equally divided to five groups according to the methods of root‐dentin pretreatment: G1: distilled water (control); G2: 2.5% NaOCl+17% EDTA; G3: MTAD; G4: PIPS; G5: MTAD+PIPS. One sample was randomly selected from each group, and scanning electron microscope (SEM) was used to observe the microscopic morphology of the coronal, middle and apical level of the root‐dentin. The remaining ten samples from each group were glued to glass fiber posts, and the bond strength was assessed by push‐out test. SEM evaluation showed that the most complete smear layer removal was found in MTAD + PIPS group, especially in coronal third. Push‐out test results exhibited that there was a statistically significant interaction between the experimental groups and root canal third (p < .0005). Significant differences could be found among five groups (p < .05): G5 > G4 > G3 > G2 > G1. G5 had the highest bond strength. In conclusion, post space pretreatments with MTAD or PIPS technique can significantly remove smear layer and improve the bond strength of glass fiber post, and combination of them works best.