Daniel J. Chiego

Autoradiographic analysis of odontoblast replacement following pulp exposure in primate teeth

Cell migration and replication associated with odontoblast replacement occurring soon after pulp exposure in primate teeth were studied. Class 5 cavity preparations resulting in pulp exposures were restored with a calcium hydroxide-containing capping agent and amalgam. Eighty-four and 96 h after this the animals were injected with 0.5 microCi/g body wt tritiated thymidine (sp. act. 6.7 Ci/mM). Teeth were extracted 6, 8, 10 and 12 days after treatment. The number of labelled cells as well as the number of grains per labelled cell were counted for odontoblast-like, fibroblast-like and perivascular cells in three 60 x 260 microns zones. These zones represented the odontoblast and cell-free (zone 1), cell-rich (zone 2) and deep pulp (zone 3) areas of normal pulp tissue. Ten sections centred around the mid-point of the exposure were counted for each tooth. Matrix formation and labelled odontoblast-like cells were observed at the interface between the capping agent and the pulp as early as day 8. Other significant findings were: (1) an increase in labelled odontoblast-like cells in zone 1 over time, suggesting a continual influx of differentiating cells; (2) an increase in labelled cells in zone 1 over time with a concurrent decrease in zone 3, suggesting that the influx of cells in zone 1 was from the deeper pulp; and (3) differences in grain counts between zones, treatment times and cell types, indicating that at least two DNA replications had occurred between initial treatment and final odontoblast-like cell differentiation.

A clinical assessment of the effects of 10% carbamide peroxide gel on human pulp tissue

Bleaching vital teeth with 10% carbamide peroxide gel is a routine procedure in which there has been no evidence of associated permanent pulpal damage. Synthesis of the enzyme heme oxygenase-1 (HO-1) is increased after exposure of eukaryotic cells to conditions of oxidative stress (including H2O2) as a defense against the damaging effects of free radicals. Dental pulps were evaluated for HO-1 (aka Heat Shock Protein 32) presence in teeth treated with 10% carbamide peroxide. Seventeen intact first premolars scheduled for orthodontic extraction were bleached for 4 h immediately preceding extraction. Fourteen additional premolars from the same individuals were not bleached. All 31 teeth were extracted, fixed, demineralized, frozen, sectioned, and immunostained with anti-HO-1 antibody using a standard ABC protocol. There was no significant difference in the presence of HO-1 between total bleached versus total unbleached teeth using the Fishers Exact Test (p < or = 0.05). However, the histological findings could be interpreted to suggest that coronal odontoblasts and endothelial cells in the underlying pulp proper may have the potential to respond to oxidative stress by increasing the synthesis of HO-1 (HSP32). This could represent a component of an initial defensive response by specific cells in strategic locations in the pulp that precedes classical inflammatory pathways.

H 3-HRP Analysis of the Nerve Supply to Primate Teeth

Sensory, sympathetic and parasympathetic ganglia located in the head and neck of rhesus monkeys were histologically examined after injection of H3-HRP into the right mandibular premolars and molars. The results showed positive labeling of ganglionic cell bodies located in the ipsilateral trigeminal, superior cervical, and otic ganglia, plus the ipsilateral mesencephalic nucleus of the trigeminal nerve.

Morphology of parasympathetic neurons innervating rat lingual salivary glands

Saliva is essential for taste function and not only does saliva influence taste reception, but also taste perception initiates salivation. As a first step in investigating circuits involved in gustatory-salivary reflexes, we have studied the morphology of the rat inferior salivatory nucleus (ISN), which contains parasympathetic secretomotor neurons that control the parotid and lingual (von Ebner) salivary glands. By applying the fluorescent label Fluorogold to the cut end of the glossopharyngeal nerve, the neurons supplying only the lingual salivary glands were labeled. Confocal microscopy and three-dimensional reconstruction were used to analyze the labeled neurons in the horizontal plane to determine their morphological characteristics. Additional neurons were studied in the coronal plane to determine the influence of the plane of section on neuron morphology. Reconstructions indicated that inferior salivatory neurons extend in a rostral-caudal distribution just adjacent to the medial border of the nucleus of the solitary tract (NST). There is considerable morphological variability among neurons, with neurons having up to 6 primary dendrites and 17 dendritic segments that extend a maximum of 834 microm from the soma. However, although ISN neurons vary in the size and complexity of their dendritic trees, distributions of all measures of neuron morphology are unimodal, indicating that distinct groups of neurons are not revealed based on these measures. There is, however, variability in the orientation pattern of the dendritic trees that is not represented in either the population or mean measures. Individual neurons can be categorized with either mediolateral, rostro-caudal or no apparent preferred orientation. Comparisons of neurons in rostral, intermediate or caudal third of the ISN revealed regional differences in neuron morphology; neurons in the caudal third have significantly longer dendrites than those in the intermediate or rostral third. Thus, while ISN neurons belong to a single morphological grouping, they vary in the size and complexity of their dendritic trees, as well as having different dendritic orientations within the salivary nucleus.

White mineral trioxide aggregate induces migration and proliferation of stem cells from the apical papilla.

INTRODUCTION Regenerative endodontic protocols recommend white mineral trioxide aggregate (WMTA) as a capping material because of its osteoinductive properties. Stem cells from the apical papilla (SCAP) are presumed to be involved in this regenerative process, but the effects of WMTA on SCAP are largely unknown. Our hypothesis was that WMTA induces proliferation and migration of SCAP. METHODS Here we used an unsorted population of SCAP (passages 3-5) characterized by high CD24, CD146, and Stro-1 expression. The effect of WMTA on SCAP migration was assessed by using transwells, and its effect on proliferation was determined by the WST-1 assay. Fetal bovine serum (FBS) and calcium chloride-enriched medium were used as positive controls. RESULTS The SCAP analyzed here showed a low percentage of STRO-1+ and CD24+ cells. Both set and unset WMTA significantly increased the short-term migration of SCAP after 6 hours (P < .05), whereas calcium chloride-enriched medium did after 24 hours of exposure. Set WMTA significantly increased proliferation on days 1-5, whereas calcium-enriched medium showed a significant increase on day 7, with a significant reduction on proliferation afterwards. SCAP migration and proliferation were significantly and steadily induced by the presence of 2% and 10% FBS. CONCLUSIONS Collectively, these data demonstrate that WMTA induced an early short-term migration and proliferation of a mixed population of stem cells from apical papilla as compared with a later and longer-term induction by calcium chloride or FBS.

Herpes simplex virus type 1 ribonucleotide reductase null mutants induce lesions in guinea pigs.

Two herpes simplex virus type 1 ribonucleotide reductase null mutants, hrR3 and ICP6 delta, produced cutaneous lesions in guinea pigs as severe as those of wild-type strains. The lesions induced by hrR3 resulted from in vivo replication of the mutant virus, suggesting that this virus-encoded enzyme is nonessential for virus replication in guinea pigs.

AUTORADIOGRAPHIC STUDY OF THE EFFECTS OF PULSED ELECTROMAGNETIC FIELDS ON BONE AND CARTILAGE GROWTH IN JUVENILE RATS

Application of pulsed electromagnetic fields (PEMF) has been used in growth and repair of non-union bone fractures. The similarities between the fibrocartilage callus in non-union bone fractures and the secondary cartilage in the mandibular condyle, both histologically and functionally, lead naturally to study the effects of PEMFs on growth in the condyle. The purposes of this study were: (1) to describe the effects of PEMFs on the growth of the condyle using autoradiography, [3H]-proline and [3H]-thymidine, and (2) to differentiate between the effects of the magnetic and electrical components of the field. Male pre-adolescent Sprague-Dawley rats (28 days old) were divided into three experimental groups of five animals each: (1) PEMF-magnetic (M), (2) PEMF-electrical (E) and (3) control, and were examined at three different times-3, 7 and 14 days of exposure. Each animal was exposed to the field for 8 h per day. Histological coronal sections were processed for quantitative autoradiography to determine the mitotic activity of the condylar cartilage and the amount of bone deposition. The PEMF (magnetic or electrical) had statistically significant effects only on the thickness of the articular zone, with the thickness in the PEMF-M group being the most reduced. Length of treatment was associated with predictable significant changes in the thickness of the condylar cartilage zones and the amount of bone deposition.(ABSTRACT TRUNCATED AT 250 WORDS)

Langerhans Cells in Apical Periodontal Cysts: An Immunohistochemical Study

The purpose of this study was to evaluate the epithelium of apical periodontal cysts for the presence of Langerhans cells using immunohistochemical techniques. Antisera to S-100 protein, muramidase, and monoclonal antibody to HLA-DR antigens were tested on formalin fixed, paraffin-embedded tissue from 60 apical periodontal cysts. S-100-positive dendritic intraepithelial cells (Langerhans cells) were found in 51 apical periodontal cysts. In 55 cases, S-100-positive cells were found in the supporting connective tissue (indeterminate cells). HLA-DR-positive intraepithelial dendritic cells were noted in 34 cases. HLA-DR also stained connective tissue macrophages. Muramidase-positive cells were found predominantly in connective tissue. Muramidase did not stain dendritic cells in the epithelium. It is concluded that Langerhans cells are present in the epithelium of apical periodontal cysts. Their function in this pathological condition is likely one of antigen processing and presentation, analogous to their role in normal epithelium. These findings provide additional support for the importance of the cell-mediated immune response in periapical disease.

Effects of denervation on 3H-fucose incorporation by odontoblasts in the mouse incisor

SummaryThe present study was designed to determine the effects of denervation on glycoprotein synthesis in the predentinal matrix of the mouse incisor. The inferior alveolar nerve (IAN), superior cervical ganglion (SCG) or both (IAN+SCG) were unilaterally resected in adult mice with the contralateral side remaining intact as a control. Fourteen days after surgery and 4 h prior to killing, 0.2 mCi of 3H-fucose was injected intravenously and mandibles were processed for standard histological and autoradiographic techniques. Silver halide grains were counted over the predentin matrix for 2000 μm per tooth. The results showed that the IAN and SCG resection affected 3H-fucose incorporation into the predentinal matrix; however, the highest absolute mean grain counts occurred after IAN+SCG resection. SCG resection increased the amount of 3H-fucose incorporated into the predentinal matrix by 48%, that of IAN by 24% and that of IAN+SCG by 14% as compared to contralateral controls. These data indicate a regulatory role for the nervous system and a possible interaction of neural components in the control of glycoprotein synthesis by odontoblasts in the mouse incisor.

Borrelidin Induces the Unfolded Protein Response in Oral Cancer Cells and Chop-Dependent Apoptosis

Oral squamous cell carcinoma (OSCC) is the most common cancer affecting the oral cavity, and US clinics will register about 30,000 new patients in 2015. Current treatment modalities include chemotherapy, surgery, and radiotherapy, which often result in astonishing disfigurement. Cancers of the head and neck display enhanced levels of glucose-regulated proteins and translation initiation factors associated with endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). Previous work demonstrated that chemically enforced UPR could overwhelm these adaptive features and selectively kill malignant cells. The threonyl-tRNA synthetase (ThRS) inhibitor borrelidin and two congeners were discovered in a cell-based chemical genomic screen. Borrelidin increased XBP1 splicing and led to accumulation of phosphorylated eIF2α and UPR-associated genes, prior to death in panel of OSCC cells. Murine embryonic fibroblasts (MEFs) null for GCN2 and PERK were less able to accumulate UPR markers and were resistant to borrelidin. This study demonstrates that UPR induction is a feature of ThRS inhibition and adds to a growing body of literature suggesting ThRS inhibitors might selectively target cancer cells.