Cement Line Staining in Undecalcified Thin Sections of Cortical Bone

A technique for demonstrating cement lines in thin, undecalcified transverse sections of cortical bone has been developed. Cortical bone samples are processed and embedded undecalcified in methyl methacrylate plastic. After sectioning at 3-5 μm, cross-sections are transferred to a glass slide and flattened for 10 min. Sections of cortical bone are stained for 20 sec free-floating in a fresh solution of 1% toluidine blue dissolved in 0.1% formic acid. The section is dehydrated in t-butyl alcohol, cleared in xylene, and mounted with Eukitt's medium. Reversal lines appear as thin, scalloped, dark blue lines against a light blue matrix, whereas bone formation arrest lines are thicker with a smooth contour. With this technique cellular detail, osteoid differentiation, and fluorochrome labels are retained. Results demonstrate the applicability of a one-step staining method for cement lines which will facilitate the assessment of bone remodeling activity in thin sections of undecalcified cortical bone.     

Cement line staining in undecalcified thin sections of cortical bone.

A technique for demonstrating cement lines in thin, undecalcified transverse sections of cortical bone has been developed. Cortical bone samples are processed and embedded undecalcified in methyl methacrylate plastic. After sectioning at 3-5 microns, cross-sections are transferred to a glass slide and flattened for 10 min. Sections of cortical bone are stained for 20 sec free-floating in a fresh solution of 1% toluidine blue dissolved in 0.1% formic acid. The section is dehydrated in t-butyl alcohol, cleared in xylene, and mounted with Eukitt's medium. Reversal lines appear as thin, scalloped, dark blue lines against a light blue matrix, whereas bone formation arrest lines are thicker with a smooth contour. With this technique cellular detail, osteoid differentiation, and fluorochrome labels are retained. Results demonstrate the applicability of a one-step staining method for cement lines which will facilitate the assessment of bone remodeling activity in thin sections of undecalcified cortical bone.     

Cement line staining in undecalcified thin sections of cortical bone

A technique for demonstrating cement lines in thin, undecalcified, transverse sections of cortical bone has been developed. Cortical bone samples are processed and embedded undecalcified in methyl methacrylate plastic. After sectioning at 3-5 microns, cross-sections are transferred to a glass slide and flattened for 10 min. Sections of cortical bone are stained for 20 sec free-floating in a fresh solution of 1% toluidine blue dissolved in 0.1% formic acid. The section is dehydrated in t-butyl alcohol, cleared in xylene, and mounted with Eukitt's medium. Reversal lines appear as thin, scalloped, dark blue lines against a light blue matrix, whereas bone formation arrest lines are thicker with a smooth contour. With this technique cellular detail, osteoid differentiation, and fluorochrome labels are retained. Results demonstrate the applicability of a one-step staining method for cement lines which will facilitate the assessment of bone remodeling activity in thin sections of undecalcified cortical bone.     

Large area sectioning for morphologic studies of nonhuman primate nasal cavities

The nasal region is important for studies in inhalation toxicology but is difficult to prepare for histological examination, especially in species as large as primates. A method for the histologic preparation of undecalcified, complete transverse sections of the nonhuman primate nasal cavity is summarized as follows. After removal of excess soft tissue, mandible and calvaria, the head is fixed in 10% neutral buffered formalin. The nasal region is transversely sectioned into serial 3-mm-thick blocks from the nares to the posterior aspect of the soft palate using a low speed saw with a water-cooled diamond-coated blade. The blocks are embedded in a mixture of glycol and methyl methacrylates, with polyethylene glycol-1500 and dibutylphthalate as plasticizers. The plastic blocks average 5.0 x 5.0 x 1.5 cm; 2-4 microns sections are cut on an automated sliding microtome. In spite of the size of the blocks, this technique yields complete transverse sections of the nasal cavities with excellent morphologic detail. The sections are amenable to a wide range of staining procedures. The procedure lends itself to autoradiographic studies. The embedding mixture is ideally suited for studies of undecalcified bone and teeth.     

Large Area Sectioning for Morphologic Studies of Nonhuman Primate Nasal Cavities

The nasal region is important for studies in inhalation toxicology but is difficult to prepare for histological examination, especially in species as large as primates. A method for the histologic preparation of undecalcified, complete transverse sections of the nonhuman primate nasal cavity is summarized as follows. After removal of excess soft tissue, mandible and calvaria, the head is fixed in 10% neutral buffered formalin. The nasal region is transversely sectioned into serial 3-mm-thick blocks from the nares to the posterior aspect of the soft palate using a low speed saw with a water-cooled diamond-coated Made. The blocks are embedded in a mixture of glycol and methyl methacrylates, with polyethylene glycol-1500 and dibutylphthalate as plasticizers. The plastic blocks average 5.0 × 5.0 × 5.1 cm; 2-4 μn sections are cut on an automated sliding microtome. In spite of the size of the blocks, this technique yields complete transverse sections of the nasal cavities with excellent morphologic detail. The sections are amenable to a wide range of staining procedures. The procedure lends itself to autoradiographic studies. The embedding mixture is ideally suited for studies of undecalcified bone and teeth.     

An Air Distributer for use in the Drop Method of Dehydration

A staining method is described using thionin, for undecalcified deacrylated bone sections. RNA is stained purplish violet, allowing still active osteoblasts to be distinguished from lining cells. Staining intensity of mineralized bone is related to the degree of mineralization. Mineralizing fronts and cement lines are visualized clearly. Lamellae show an alternate pattern. Histomorphometric parameters such as osteon thickness and interstitial bone thickness can be measured without using polarized light. The mineralizing front can be assessed and expressed as a percentage of the osteoblast-covered interface between osteoid and mineralized bone. The stain is also useful for qualitative assessment of metabolic bone disease. Thionin stained sections can be kept for at least one year when stored hi the dark at 7 C.     

Dentition and age determination of the giraffe Giraffa camelopardalis

Thirteen tooth eruption stages and their corresponding chronological ages are descri bed from a series of giraffe jaws. These can be used for age determination in giraffes with immature dentition. Significant correlations of the lingual crown height ( r =0-957; P < 0001) and lingual occlusal surface width ( r =0-959; P < 0001) with the number of dark staining incremental lines in the cementum of thin decalcified sections of the maxillary first molar were found. The regression equations derived from these relationships provide a further method for determining the age of a giraffe. A composite plate showing maxillary first molar wear patterns provides a means of roughly assigning an age to a particular specimen. Thin sections of undecalcified teeth, mandible measurements, various other indices of tooth wear and eye lens mass were investigated and found unsuitable for age determination in this     

The Enamel Microstructure of the Early Eocene Pantodont Coryphodonand the Nature of the Zigzag Enamel

An additional method for the investigation of the microstructure of enamel is described using the teeth of Coryphodon, Uintatherium, Entelodon, and Crocuta. Under low magnification natural surfaces or sections of teeth display details of the enamel microstructure when the light guide effect of prisms is used. Under the same low magnification even more details were obtained from sputter-coated surfaces of sections. The method is of particular significance for the investigation of large teeth with thick enamels when structures are somewhat irregular. The new method provides a better general survey, where scanning electron microscope images often show confusing details. The enamel of Coryphodon shows oblique lines of nested chevrons that are similar, to some degree, to the zigzag enamel in Crocuta, but a distinct asymmetry between ascending and descending lineaments was observed. This specific Coryphodon -enamel was also found in Uintatherium and Entelodon. This enamel type, which evolved several times in parallel, cannot be attributed to a specific diet, but must be regarded as one of the several ways to strengthen the enamel against breakage.     

Evaluating the effectiveness of teeth and dorsal fin spines for non‐lethal age estimation of a tropical reef fish,coral trout Plectropomus leopardus

This study investigated whether teeth and dorsal fin spines could be used as non‐lethal methods of age estimation for a vulnerable and highly valued tropical fisheries species, coral trout Plectropomus leopardus. Age estimation of individuals from 2 to 9 years old revealed that dorsal spines represent an accurate ageing method (90% agreement with otoliths) that was more precise [average per cent error (APE) = 4·1, coefficient of variation (c.v .) = 5·8%] than otoliths (APE = 6·2, c.v . = 8·7%). Of the three methods for age estimation (otoliths, dorsal spines and teeth), spines were the most time and cost efficient. An aquarium‐based study also found that removing a dorsal spine or tooth did not affect survivorship or growth of P. leopardus. No annuli were visible in teeth despite taking transverse and longitudinal sections throughout the tooth and trialling several different laboratory methods. Although teeth may not be suitable for estimating age of P. leopardus, dorsal spines appear to be an acceptably accurate, precise and efficient method for non‐lethal ageing of individuals from 2 to 9 years old in this tropical species.     

Age related changes in ovarian follicular kinetics in the Indian skipper frogRana cyanophlyctis (Schn.)

Changes in ovarian follicular kinetics were studied in relation to aging in the Indian skipper frog Rana cyanophlyctis.Age was determined by skeletochronology, by counting the number of growth rings and lines of arrest of growth from the cross sections of 4th phalange of 4th toe. For follicular kinetics study oocytes were counted under binocular using 10% of Bouin’s fixed ovary and they were classified into first growth phase, medium-sized second growth phase, large-sized second growth phase and atretic follicles. Analysis of phalangeal cross sections indicated that frogs ranging 14–54 g in body weight and 4.9–8.9 cm in body size showed 1–7 year rings. Frogs that weighed 14–16 g showed 1 year ring, and contained immature ovaries; those with 18 g body weight had one to two year rings, in which second growth phase oocytes appeared for the first time in the primiparous ovary. Frogs with 20–54 g body weight showed 2–5 year rings in which ovary contained 5–24% of second growth phase oocytes. Further, body weight, body size, ovarian weight, number and size of second growth phase oocytes and total number of oocytes showed a significant (P < 0.05) positive correlation, while, the number of first growth phase and atretic follicles showed a poor correlation with age. The results suggest that in nature, the age of Rana cyanophlyctis ranges between 1–7 years. Phalangeal growth rings are formed annually. Females attain sexual maturity in 2nd year. Frogs with 2–5 years of age may constitute breeding females. Body weight, body size, ovarian mass, number of second growth phase and total oocytes, and egg size increase with age up to 5 years.     

Growth layers in dental cementum ofSaguinus monkeys in South America

Dark stained layers were observed in the cementum of the teeth ofSaguinus monkeys living in South America. Since the number of layers correlated with the degree of attrition in the incisor, the dark layers probably showed the growth lines. The teeth of the monkeys captured at the beginning of the wet season showed the incremental dark line around most of the exterior of the cementum.     

A Method for Histological Examination of Undecalcified Teeth

A method is presented for histological examination of undecalcified ground sections of tooth roots affected with periodontal disease. The roots were placed in Karnovsky's fixative overnight, postfixed in 2% buffered osmic acid, and dehydrated in an ascending series of ethanol. The specimens were then infiltrated with propylene-oxide and Epon-Araldite resin, embedded in Epon-Araldite, and sections were prepared using a cutting and grinding system. The resulting ground sections were 8-12 μm thick. The sections were allowed to air dry at room temperature. When thoroughly dried, a coverglass was applied using resinous mounting medium DPX. The specimens were examined by phase-contrast microscopy. The method is useful for simultaneous examination of mineralized dental tissue and bacterial morphotypes covering the root surface of teeth involved with periodontal disease.     

A Method for Histological Examination of Undecalcified Teeth

A method is presented for histological examination of undecalcified ground sections of tooth roots affected with periodontal disease. The roots were placed in Karnovsky's fixative overnight, postfixed in 2% buffered osmic acid, and dehydrated in an ascending series of ethanol. The specimens were then infiltrated with propylene-oxide and Epon-Araldite resin, embedded in Epon-Araldite, and sections were prepared using a cutting and grinding system. The resulting ground sections were 8-12 μm thick. The sections were allowed to air dry at room temperature. When thoroughly dried, a coverglass was applied using resinous mounting medium DPX. The specimens were examined by phase-contrast microscopy. The method is useful for simultaneous examination of mineralized dental tissue and bacterial morphotypes covering the root surface of teeth involved with periodontal disease.     

Histological Observations of Dental Tissues Using the Confocal Laser Scanning Microscope

To investigate the time course of mineralization in undecalcified dental tissues, calcein-and tetracycline-labeled rat maxillary molar sections were stained with Villanueva bone stain en bloc, embedded in methyl-methacrylate (MMA), ground to 50 μm thickness, and observed by confocal laser scanning microscopy (CLSM). This method allowed observation of dental structures including odontoblasts, pulp cells and periodontal ligament, and dentinal tubules and enamel rods at high resolution; labeled enamel, dentine, and cementum could be observed simultaneously regardless of section thickness. CLSM permitted simultaneous observation of both the components of calcified tissue and the cellular components of dental tissues, and assessment of the mineralization time course of hard tissues labeled by tetracycline or calcein. The technique is useful for both assessing the elements composing dental structure and observing the histological dynamics by which dental structure develops.     

Investigating prevalence of dental anomalies in Eastern Province of Saudi Arabia through digital orthopantomogram

ObjectiveThe aim of this study was to investigate the prevalence of dental anomalies in the Eastern Province of Saudi Arabia using the digital Orthopantomography (OPG).MethodsA retrospective radiographic study was performed in which digital OPGs of 1189 subjects, ages ranging between 7 and 65 years were reviewed, and 1104 fulfilled inclusion criteria. Statistical analysis was performed. The OPGs were reviewed for congenitally missing teeth, impactions, ectopic eruption, supernumerary teeth, odontomas, dilacerations, taurodontism, dens in dente, gemination and fusion.ResultsOPGs of 1104 patients with mean age 35.32 ± 16.63 were included. The total prevalence of developmental anomalies in this study was 36.3% (401/1104). Male and female subjects with anomalies were 133 (33.2%) and 268 (66.8%) respectively. The prevalence of dilacerated teeth 300 (30.2%), congenitally missing teeth was 246 (24.7%), supernumerary teeth 18 (1.8%), talon cusp and taurodontism were seen in one patient each 1 (0.1%). Of these, a total of 15 (1.5%) anomalies were noted in pediatric patients.ConclusionsDilaceration was the most prevalent anomaly (30.2%) in the studied sample followed by congenitally missing teeth (23.4%). Talon cusp, concrescence/fusion, and taurodontism were the least prevalent anomalies.     

Examination of the Relationship between Oral Health and Arterial Sclerosis without Genetic Confounding through the Study of Older Japanese Twins

ObjectiveAlthough researchers have recently demonstrated a relationship between oral health and arterial sclerosis, the genetic contribution to this relationship has been ignored even though genetic factors are expected to have some effect on various diseases. The aim of this study was to evaluate oral health as a significant risk factor related to arterial sclerosis after eliminating genetic confounding through study of older Japanese twins.ResultsWe examined 91 monozygotic and 15 dizygotic twin pairs (males: 42, females: 64) with a mean (± standard deviation) age of 67.4 ± 10.0 years. Out of all of the oral health-related measurements collected, only the number of teeth was significantly related to arterial sclerosis (odds ratio: 0.72, 95% confidence interval: 0.52-0.99 per five teeth). Regression analysis showed a significant association between the IMT-Cmax and the number of teeth as an environmental factor (p = 0.037).ConclusionsAnalysis of monozygotic twins older than 50 years of age showed that having fewer teeth could be a significant environmental factor related to arterial sclerosis, even after controlling for genetic and familial confounding.     

Gastric teeth of some thoracotreme crabs and their contribution to the brachyuran phylogeny

The gastric teeth of three ocypodoid species were investigated using scanning electron microscopy, and the morphological results were discussed with respect to the known food preferences. The species were chosen in particular because of contrasting ideas about their relationships within the Thoracotremata. For the genera Heloecius, Dotilla, Mictyris, and “Uca” (s. str.), we find a specific correlation of the gastric teeth with the suspension feeding. The lateral gastric teeth of Uca have no prominent lateral teeth cusps, and most of their teeth surface consists of transverse comb‐like lamellae. However, this possible food adaptation does not exclude the usability of specific teeth characters to distinguish species of suspension feeders. The closer relationship of the Dotillidae to grapsoid lines of gecarcinid or sesarmid crabs suggested by molecular data is not supported by the gastric teeth. For the genus Ucides, we found several characters that distinguish Ucides from the remaining ocypodoid genera Heloecius, Dotilla, Mictyris, and “Uca.” In particular, the structures of the lateral and the dorsomedian teeth show some similarities to genera of the Gecarcinidae and Sesarmidae. Our results suggest that foregut characters can be used for phylogenetic analyses. J. Morphol. 2011. © 2011 Wiley‐Liss, Inc.     

Bromodeoxyuridine (BrdUrd) immunohistochemistry in undecalcified plastic-embedded tissue. Elimination of the DNA denaturation step

Summary We examined the application of BrdUrd immunohistochemistry to detect S-phase cells in undecalcified bone and cartilage from the growing rat embedded in Spurr's resin. The effect of fixation on the procedure was studied, and the validity of the technique examined by a comparative study with tritiated thymidine ([³H]-TdR) autoradiography. The use of sodium-ethoxide to remove plastic from tissue sections prior to immunohistochemistry resulted in the production of sufficient ssDNA to make a separate DNA denaturation step unnecessary, thus sparing sections from potentially destructive treatment and shortening the immunohistochemical procedure. Fixation in formalin or Bouin's fluid gave the most satisfactory results. The distribution of BrdUrd labeled cells was restricted to the sites of cell proliferation in growing long bones. Combined studies with BrdUrd immunohistochemistry and [³H]-TdR autoradiography showed that the majority of BrdUrd labeled cells had also incorporated [³H]-TdR, thus attesting to the validity of the technique. This novel approach is suitable for the study of undecalcified hard tissues as well as soft tissues.     

Oriented spermatozoa in the spermatophore of the palaemonid shrimp, Macrobrachium rosenbergii

Estimates of the degree of orientation of spermatozoa within spermatophores of the freshwater shrimp, Macrobrachium rosenbergii, were made using electron micrographs of ultrathin sections cut transverse and parallel to the longitudinal axis of the spermatophores. Counts of the total number of longitudinal and non-longitudinal profiles of sperm spikes in transverse sections of spermatophores indicated that 67% of the spermatozoa were oriented with their spikes approximately perpendicular to the long axis of the spermatophore. In longitudinal sections, ~30% of the sperm spike profiles were oriented parallel to the longitudinal axis of the spermatophore, leaving ~70% of the profiles oriented in planes not parallel to the longitudinal spermatophore axis. Stereologic analyses based on the number of intersections of longitudinally sectioned sperm spike profiles per unit length of test lines placed over photographic images of ultrathin sections cut in both transverse and longitudinal planes of spermatophores gave a Saltykov approximation of 0.607, favoring the view that the spermatozoa were oriented with their spikes perpendicular to the long axis of the spermatophore. This orientation brings many of the sperm bases approximately parallel to the surface of the spermatophore, an orientation which may facilitate normal sperm-egg interaction during fertilization in this species.