This review focuses on the process of enamel maturation, a series of events associated with slow, progressive growth in the width and thickness of apatitic crystals. This developmental step causes gradual physical hardening and transformation of soft, newly formed enamel into one of the most durable mineralized tissues produced biologically. Enamel is the secretory product of specialized epithelial cells, the ameloblasts, which make this covering on the crowns of teeth in two steps. First, they roughly "map out" the location and limits (overall thickness) of the entire extracellular layer as a protein-rich, acellular, and avascular matrix filled with thin, ribbon-like crystals of carbonated hydroxyapatite. These initial crystals are organized spatially into rod and interrod territories as they form, and rod crystals are lengthened by Tomes' processes in tandem with appositional movement of ameloblasts away from the dentin surface. Once the full thickness of enamel has been formed, ameloblasts initiate a series of repetitive morphological changes at the enamel surface in which tight junctions and deep membrane infoldings periodically appear (ruffle-ended), then disappear for short intervals (smooth-ended), from the apical ends of the cells. As this happens, the enamel covered by these cells changes rhythmically in net pH from mildly acidic (ruffle-ended) to near-physiologic (smooth-ended) as mineral crystals slowly expand into the "spaces" (volume) formerly occupied by matrix proteins and water. Matrix proteins are processed and degraded by proteinases throughout amelogenesis, but they undergo more rapid destruction once ameloblast modulation begins. Ruffle-ended ameloblasts appear to function primarily as a regulatory and transport epithelium for controlling the movement of calcium and other ions such as bicarbonate into enamel to maintain buffering capacity and driving forces optimized for surface crystal growth. The reason ruffle-ended ameloblasts become smooth-ended periodically is unknown, although this event seems to be crucial for sustaining long-term crystal growth.

Cellular and Chemical Events During Enamel Maturation

5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazolyl)-3-(4-sulfophenyl)tetrazolium, inner salt (MTS) and related analogs of 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide (MTT) reducing to purple water-soluble formazans As cell-viability indicators

The application of a modified colorimetric bioassay for the evaluation of the biological effects of mycotoxins is reported. Using three different monolayer cell lines (swine kidney, Madin Darby canine kidney, HeLa) the influence of nine different mycotoxins on the cellular methylthiazoltetrazolium (MTT)-cleavage activity was evaluated. The yellow tetrazolium salt MTT is converted by mitochondrial dehydrogenases of metabolically active cells to an insoluble purple formazan product, which was then solubilized with dimethylsulfoxide. The optical density of this homogeneous solution was suitable for a precise spectrophotometric measurement by a plate reader at a wavelength of 510 nm. Nine mycotoxins were simultaneously tested in all three cell lines, from which the swine kidney cell line proved to be the most sensitive. The effects of additional 35 mycotoxins were therefore tested using swine kidney monolayers as target cells. A total of 28 toxins of the 44 mycotoxins tested proved to be cytotoxic in the MTT-bioassay. Most of them belong to the group of trichothecene mycotoxins. Concentrations ranged between 0.01 µg and 100 µg/ml of cell culture medium. The MTT cleavage assay was found to be a quick (24 hours) and easy to perform system for the evaluation of the biological activity of many different mycotoxins and may also provide a useful tool for the testing of a large variety of sample materials.

Cytotoxicity of mycotoxins evaluated by the MTT-cell culture assay.

Effects of surface roughness of titanium implants on bone remodeling activity of femur in rabbits.

The osteoclast is distinguished from other macrophage polykaryons by its polarization, a feature induced by substrate recognition. The most striking component of the polarized osteoclast is its ruffled membrane, probably reflecting insertion of intracellular vesicles into the bone apposed plasmalemma. The failure of osteoclasts in c-src−/− osteopetrotic mice to form ruffled membranes indicates pp60c-src (c-src) is essential to osteoclast polarization. Interestingly, c-src itself is a vesicular protein that targets the ruffled membrane. This being the case, we hypothesized that matrix recognition by osteoclasts, and their precursors, induces c-src to associate with microtubules that traffic proteins to the cell surface. We find abundant c-src associates with tubulin immunoprecipitated from avian marrow macrophages (osteoclast precursors) maintained in the adherent, but not nonadherent, state. Since the two proteins colocalize only within adherent avian osteoclast-like cells examined by double antibody immunoconfocal microscopy, c-src/tubulin association reflects an authentic intracellular event. C-src/tubulin association is evident within 90 min of cell-substrate recognition, and the event does not reflect increased expression of either protein. In vitro kinase assay demonstrates tubulin-associated c-src is enzymatically active, phosphorylating itself as well as exogenous substrate. The increase in microtubule-associated kinase activity attending adhesion mirrors tubulin-bound c-src and does not reflect enhanced specific activity. The fact that microtubule-dissociating drugs, as well as cold, prevent adherence-induced c-src/tubulin association indicates the protooncogene complexes primarily, if not exclusively, with polymerized tubulin. Association of the two proteins does not depend upon protein tyrosine phosphorylation and is substrate specific, as it is induced by vitronectin and fibronectin but not type 1 collagen. Finally, consistent with cotransport of c-src and the osteoclast vacuolar proton pump to the polarized plasmalemma, the H+-ATPase decorates microtubules in a manner similar to the protooncogene, specifically coimmunoprecipitates with c-src from the osteoclast light Golgi membrane fraction, and is present, with c-src, in preparations enriched with acidifying vesicles reconstituted from the osteoclast ruffled membrane.

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Substrate Recognition by Osteoclast Precursors Induces C-src/Microtubule Association

A simple in vito cytotoxicity test using the MTT (3-(4,5)-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) colorimetric assay: analysis of eugenol toxicity on dental pulp cells (RPC-C2A).

Toxicity of camphorated phenol and camphorated parachlorophenol in dental pulp cell culture.

The effects of colchicine or vinblastine on the blood calcium level in rats

An autoradiographic study on rats was carried out with 55Fe and colchicine in order to investigate the Arole of microtubules in the iron transport process from rat incisor ameloblasts to the enamel surface. In the control rats injected with 55Fe, autoradiographic silver grains were distributed over the ameloblasts during the whole stage of enamel maturation and over the enamel surface at the enamel pigmentation stage. After the colchicine injection, the area of ameloblasts with smooth distal cell membranes increased in the maturation stage. These ameloblasts were heavily injured, and the iron-containing pigment was released from the cell. The number of silver grains decreased in these ameloblasts. However, the silver grains increased in the area of ameloblasts with ruffled distal cell membranes. At the enamel pigmentation stage, the silver grains over the ameloblasts increased, and those over the enamel surface were reduced. The accumulation and dislocation of the iron-containing pigment was noted in the ...

Effect of colchicine on iron transport during stages of maturation and enamel pigmentation in rat incisor enamel: an autoradiographic study using 55Fe.

The present study was carried out to investigate the alveolar bone resorption in the molar tooth region in the rats fed a low calcium diet. Male Wistar rats (70-85g in body weight) were either fed a low calcium diet (0.05% Ca, 0.35% P) or a control diet (0.5 % Ca, 0.35% P) by using a pair feeding technique. The rats were sacrificed at intervals of 3, 6, 9 and 20 days during the experimental period. Contact microradiograph of the second molar region of the mandible showed that the cancellus bone as well as the endosteal surface of the cortical bone were progressively resorbed soon after the start of low calcium feeding. At day 9, the amount of bone was reduced to about half of the control rats and 80% of the bone was diminished at day 20. In spite of the severe bone resorption, the alveolar bone proper that surrounds the molar sockets and a few cancellus bone were seen remaining and the occlusal function of the molar tooth seemed to be maintained. These results suggest that the alveolar bone quickly responded to the low calcium diet resulting in bone resorption and that the bone in the supporting tissues of the molar tooth seems to be not affected by the calcium deficiency.

https://www.jstage.jst.go.jp/article/koubyou1952/59/3/59_3_585/_pdf

Hideaki Ogura

Papers

A simple in vito cytotoxicity test using the MTT (3-(4,5)-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) colorimetric assay: analysis of eugenol toxicity on dental pulp cells (RPC-C2A).

Toxicity of camphorated phenol and camphorated parachlorophenol in dental pulp cell culture.

The effects of colchicine or vinblastine on the blood calcium level in rats

Effect of colchicine on iron transport during stages of maturation and enamel pigmentation in rat incisor enamel: an autoradiographic study using 55Fe.

[Alveolar bone resorption process in molar tooth region in calcium-deficient rats].