Influence of various forms of fixation and decalcification on immunohistochemical staining of paraffin-embedded human teeth and surrounding tissues was initially examined using commercially available antibodies against vimentin (V), type I collagen (C) and cytokeratin (K). Secondly, monoclonal antibody (MoAb) was produced against both bovine and human cementum and immunohistochemical screening was subsequently undertaken to test reactivity with different scheme of fixation and decalcification. The combination of neutral buffered paraformaldehyde fixation, Morse solution and unmasking procedure yielded both optimal morphology and immunoreactivity. The application of this method into production of MoAb against human or bovine cementum generated a variety of MoAbs reactive with both human teeth and surrounding tissues. The percentage of hybridoma supernatant reactive with sections of human teeth was 14.0-22.4% and was both higher and much improved compared to results of previous soft tissue studies. Finally, the MoAbs, BC 1 and 2 (isolated following the use of bovine cementum as immunogen) and HC 1 (human cementum immunogen) recognized specific bands of various molecular weight. The three MoAbs showed strong resistance against periodate oxidation and borohydrate reduction and were stable following treatment with proteolytic enzymes. All were immunoreactive with components of cementum.

View full abstract

Some prior reports have suggested that guided tissue regeneration (GTR) procedures achieve only partial regeneration and induces the ankylosis rather than true attachment. Accordingly, others have developed an alternative procedure employing gelatine membrane compounded with bovine cementum particles (CGM) which has proven effective in stimulating a more physiologic form of attachment. This study was undertaken to perform a direct comparison of histological results when CGM and GTR membrane were used at comparable sites in the same monkey. Three monkeys with no periodontal disease were used. Following flap surgery, recession type defects were created on the buccal side of the maxillary lateral incisors and second premolars, and the cementum was removed from the root surface at an area corresponding to the bone crest. The right and left lateral incisors and second premolars were covered with CGM and GTR membrane, respectively. The GTR membranes were removed after 4 weeks. At 6 wks, the animals were sacrificed, and specimens were prepared for histological examination. More coronally placed true new attachment was observed following application of CGM to the planed root surfaces. Application of the GTR membrane resulted in formation of bonelike cementum and ankylosis, whereas CGM established true periodontal regeneration.

View full abstract

We investigated biochemically and immunohistochemically the age-related changes in collagen (types I, III and IV), laminin and tenascin of the extracellular matrix in the submandibular gland of infant rats. The hydroxyproline content markedly increased on day 14 after birth. Electrophoretic profile revealed α chains of only type I collagen up to day six after birth. Type III collagen, which was diffusely distributed in interlobular connective tissue, was gradually expressed from day seven after birth. Type IV collagen content increased from day seven. Laminin content gradually increased with age. Tenascin immunohistochemically showed weak expression only around the vessel walls up to day seven. In addition, this expression markedly increased around acinar and ductal walls as basement membrane components on day 14 after birth.
These findings suggest that rat submandibular gland types I and IV collagen, and laminin are already present during the fetal stage and relate to construction of tissue, and that type III collagen and tenascin, which increased from day seven after birth, relate to maturation.

View full abstract

The effects of PBS heat fixation on tissue structure, immunostaining, and in situ RT-PCR were investigated using excised mucosal tissues from both humans and various animals. It was found that basal cells were stretched to two to three times their normal length at 60℃, and 4 to 5 times at 90℃, resulting in detachment of the epithelium and basal cells from subjacent connective tissue. Tonofibrils disappeared completely and became an electron dense amorphous mass at 60℃. Both nuclear and cytoplasmic membranes also disappeared as did intracytoplasmic organelles. Electron dense amorphous-material occupied the entire nucleus with fixation at 90℃. Collagen fibrils appeared swollen at 60℃ and either disappeared completely or became fragmented undergoing transition to an electron dense amorphous conglomerates at 90℃.
Immunostaining of cytokines disappeared at 60℃, and that of vimentin at 90℃. However, cytokeratin and human leukocyte common antigen (CD 45RB) reactivity remained intact at both temperatures. IL-6 mRNA could be localized by in situ RT-PCR even after thermal fixation.
Results suggest that a proper combination of immunohistochemistry and in situ RT-PCR will enable investigators to localize proteins in addition to DNA or RNA within oral tissue sections. This capability should prove helpful in applied histopathologic diagnostic techniques.

View full abstract

Light and electron microscopic investigations of lectin binding patterns in rat tooth germs were undertaken in order to elucidate glycoconjugate localization in cells of the reduced enamel epithelia and their derivatives. It was found that Ulex europaeus agglutinin (UEA-1), peanut agglutinin (PNA), soybean agglutinin (SBA), and wheat germ agglutinin (WGA) exhibited variable reactivity patterns with different epithelia. UEA-1 was reactive with cells of the stratum intermedium and stellate reticulum in the tooth germ but unreactive with ameloblasts, outer enamel epithelial cells, and junctional epithelium at later stages. Reaction patterns of PNA in these cells differed from those of UEA-1. Results indicated that inner and outer cells of the reduced enamel epithelium are heterogeneous with regard to lectin binding patterns.

View full abstract

Although diabetes mellitus is known to aggravate periodontal disease, the precise relationship between these two entities is far from being completely understood. Further study of this relationship was therefore undertaken in the form of observation of both naturally occurring gingivitis in rats (ODUS/Odu) and effects produced by induction of experimental diabetes mellitus by injection of streptozotocin (STZ: 65mg/kg, i.v.). At one and 3 mon after STZ injection, liquid paraffin was injected intraperitoneally. Four days thereafter, pocket probing depths of rats were measured and blood samples as well as peritoneal macrophages were collected from both experimental animals and non diabetic controls. Both chemotaxis and phagocytosis of macrophages were studied. At one and 3 mon after STZ injection, pocket probing depths of diabetic animals were significantly deeper than those of controls (p<0.001). Pocket probing depths were deeper at 3 mon after STZ injection than after 1 mon in diabetic animals. At three months after STZ injection, there was a high degree of positive correlation between pocket probing depths, blood glucose levels, triglyceride, and hemoglobin A_1c levels (p<0.01). Also, macrophage chemotaxis was more suppressed in diabetic rats than it was in controls. Additionally, both phagocytosis ratios and phagocytosis indices of macrophages in the diabetes group were significantly more suppressed than those in controls in both experimental periods (p<0.001). Findings suggest that both chemotaxis and phagocytosis are compromised in macrophages from rats rendered diabetic by STZ injection. Thus as host defense mechanisms become weakened, there is a corresponding progression of periodontal disease.

View full abstract

The medulla oblongata caudal to the obex was explored for neurons responsive to tooth pulp (TP) stimulation in cats. Four different subclasses of TP neurons were found. The latter included TP specific (TPS) neurons, trigeminal wide dynamic range (trigeminal WDR) neurons with TP input, trigeminal subnucleus reticularis ventralis (trigeminal SRV) neurons with TP input and convergent reticular formation (convergent RF) neurons with TP input. TPS neurons were located in the dorsal marginal rim of the trigeminal subnucleus caudalis, i.e., in the marginal layer or the outer zone of substantia gelatinosa. WDR neurons with TP input were found in the neck region of medullary dorsal horn which corresponds to the lateral part of subnucleus reticularis dorsalis (SRD). Trigeminal SRV neurons with TP input were located in the lateral part of SRV. Convergent RF neurons with TP input were found in the middle third of the caudal bulbar RF consisting of SRD and SRV. Both TPS neurons and WDR neurons with TP input included trigeminothalamic neurons as evidenced by the antidromic activation from the nucleus ventralis posteromedialis of the contralateral thalamus. A significant proportion of both trigeminal SRV and convergent RF neurons with TP input were antidromically activated by stimulation of the nucleus centralis lateralis of the contralateral thalamus. The former two subclasses may subserve the sensory-discriminative aspect of toothache, while the latter two subclasses, the emotional-motivational aspect.

View full abstract

We investigated the use of India ink as an indicator of root canal sealing ability. Sealing ability is one of the most important factors required of root canal cements. Various dyes have been used for this purpose. Methylene blue, radioisotopes and India ink have all been tried. However, there is no evidence that India ink is suitable as an indicator. We measured the particle size distribution of India ink to determine how this factor affects test results. In addition, we observed the surface texture of an experimentally developed calcium phosphate sealer and a commercially available root canal sealer using SEM. We found that a portion of the India ink particles were smaller than cracks on the two sealer surfaces, indicating, that India ink could pass through these cracks. We concluded that India ink is suitable as an indicator of root canal seal.

View full abstract