Chi-Chuan Hsieh

Cytotoxicity of sodium fluoride on human oral mucosal fibroblasts and its mechanisms

Because sodium fluoride (NaF) is widely used for prevention of dental caries, pathobiological effects of NaF were investigated on human oral mucosal fibroblasts. The results showed that NaF was cytotoxic to oral mucosal fibroblasts at concentrations of 4 mmol/L or higher. Exposure of cells to NaF for 2 h also inhibited protein synthesis, cellular ATP level and functional mitochondrial activities in a dose-dependent manner. However, incubation of cells with NaF up to 12 mmol/L for 2 h depleted only 13% of cellular glutathione level. The IC50 of NaF on cellular ATP level was about 5.75 mmol/L. Preincubation of the cells with pyruvate and succinate did not protect cells from NaF-induced ATP depletion. At concentrations of 4 mmol/L, 8 mmol/L and 12 mmol/L, NaF inhibited 31%, 56% and 57% of mitochondrial functions, respectively, after 2 h incubation. No significant inhibition for NaF was found at concentrations lower than 2 mmol/L (40 ppm). These results indicate that NaF can be toxic to oral mucosal fibroblasts in vitro by its inhibition of protein synthesis, mitochondrial function and depletion of cellular ATP. Because of repeated and long-term usage of NaF, more detailed studies should be undertaken to understand its toxic effects in vitro and in vivo.

Collagen Biosynthesis in Human Oral Submucous Fibrosis Fibroblast Cultures

To investigate the mechanism of collagen accumulation in oral submucous fibrosis (OSF) tissues, we examined the biosynthesis of collagen in fibroblast cultures established from OSF lesions. Fibroblasts obtained from four of ten OSF specimens showed more than a 1.5-fold increase in the production of collagens compared with fibroblasts from age-, sex-, and passage-matched normal controls (p < 0.05). When the relative amounts of collagen synthesis were estimated by SDS polyacrylamide gel electrophoresis, it was found that both OSF and control cells produced about 85% type I collagen and 15% type III collagen. The ratio of α1(I) to a2(I) chains was about 3:1 in OSF cells instead of the 2:1 expected for type I collagen. The excess al(I) chains could mean that collagen type I trimer was synthesized by the fibroblasts. These findings suggest that collagen overproduction and a reduced degradation of the structure-stable collagen type I trimer synthesized by OSF fibroblasts might contribute to the accumulation of collagen in OSF lesions in vivo. The mechanism(s) of increased procollagen production were analyzed by Northern blot, slot blot, and Southern blot. The OSF fibroblast strains with elevated collagen production also contained higher-than-normal levels of procollagen mRNA, and the ratios of α1(I), a2(I), and α1(III) procollagen mRNAs were compatible with the results of corresponding procollagen a chains. The gene copy number of proa2(I) collagen gene in OSF fibroblasts was about 1.05. No gene amplification was found. These results indicate that expression of these procollagen genes in cultured fibroblasts is regulated at the transcriptional level.

Apatitic Fluoride Increase in Enamel from a Topical Treatment Involving Intermediate CaHPO4·2H2O Formation, an in vivo Study

Previous laboratory and animal studies have reported high levels of apatitic fluoride uptake from a topical fluoride procedure in which teeth were pretreated with an acidic solution saturated with respect to CaHPO4 • 2H2O (DCPD) prior to the fluoride treatment. A study was initiated to determine the fluoride uptake from this procedure under clinical conditions. A microbiopsy technique was used to measure enamel fluoride contents before, 1 month after, and 3 months after an application of an acidulated phosphate fluoride (APF) solution alone or in combination with a DCPD pretreatment on the maxillary incisors of 50 fourth-grade children in a nonfluoride area. 1 month after treatment, there was no significant difference in enamel fluoride concentration between APF and control groups. The DCPD/APF treatment produced mean fluoride uptakes of 1,011 ± 134 ppm (n = 50) and 2,042 ± 221 ppm (n = 47) in the outer 3.5 μm of enamel when measured 1 and 3 months, respectively, after treatment. The results indicate that the DCPD pretreatment can substantially increase permanently bound fluoride uptake under clinical conditions.

Morphological alterations associated with the cytotoxic and cytostatic effects of citric acid on cultured human dental pulp cells

Citric acid exerts potential harmful effects on the pulp when used for root surface demineralization, smear layer removal, and dentin etching. Using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay, we found that incubation of cultured human dental pulp cells in medium containing 0.5% (pH 4.74) or 1.0% (pH 3.42) of citric acid for 2 h lead to 25% and 48% of cell death, respectively. Cytotoxicity of citric acid was associated with its acidity. Exposure of cells to pure 1% citric acid (pH 2.26) for 60 s lead to immediate cell death. Cytotoxicity was usually preceded by cell retraction, cell surface blebbing, and finally uptake of trypan blue, implicating the presence of cell membrane damage. A medium containing 0.05% citric acid can retard the growth of pulp cells. These results indicate that adequate protection of the pulp is important, especially when the remaining dentin is thin in deep carious lesions or in the presence of accessory canals on the root surface.

Thrombin-induced DNA synthesis of cultured human dental pulp cells is dependent on its proteolytic activity and modulated by prostaglandin E2*

To clarify the roles of alpha-thrombin and prostaglandin E2 (PGE2) in the healing and inflammatory processes of dental pulp, their effects on the DNA synthesis of human pulp cells were investigated by measurement of [3H]thymidine incorporation. At a concentration range of 1 to 25 units/ml, alpha-thrombin stimulated DNA synthesis of the pulp cells by 1.5 to 2.6-fold. On the contrary, PGE2 (> 0.05 microgram/ml) suppressed DNA synthesis by 24 to 39%. Using reverse transcriptase-polymerase chain reaction, thrombin receptor mRNA expression was identified in the pulp cells. Furthermore, alpha-thrombin-induced DNA synthesis could be inhibited by antithrombin III (2 units/ml) with heparin (2 units/ml) or D-Phe-Pro-ArgCH2Cl (50 micrograms/ml). PGE2 (0.1 to 0.5 microgram/ml) also inhibited the thrombin-induced DNA synthesis by 39 to 64%. These results imply that pulp cells express the thrombin receptor that is activated by the serine protease activity of thrombin. Interactions of thrombin and PGE2 are important in modulating the inflammatory and healing processes of the pulp.

Root deformation during root-end preparation

Ultrasonic root-end preparation techniques have recently been introduced and revolutionized the field of endodontic surgery. However, several reports claimed that there was an increasing incidence of crack formation after ultrasonic root-end preparation. As yet, little work has focused on the root deformation during root-end preparation. Thus, the purpose of this investigation was to measure the amount of root deformation during root-end preparation with the use of microhandpiece and ultrasonic systems by using strain gauge methods, and simultaneously to detect any cracks with the aid of the stereomicroscope, stain, and an image processing system. The results demonstrated the ultrasonic instrumentation produced significantly greater strain on average than that generated with the microhandpiece system. From the viewpoint of fracture, any technique that could diminish the strain on the root would decrease the likelihood of fracture; however, no crack was observed on any resected surface of roots in this study.

Thrombin activates the growth, cell-cycle kinetics, and clustering of human dental pulp cells

Thrombin is activated during vascular injury and inflammation of the dental pulp. In the present study, we found that thrombin can stimulate the proliferation of pulp cells in a dose-dependent manner as analyzed by modified MTT assay. The cell number increased by 1.6, 1.77, and 2.14-fold over that of control after exposure to 5, 10, and 20 units/ml of thrombin for 5 days. Flow cytometry studies also found that thrombin (10 units/ml) can induce the cell cycle progression of pulp cells after 24 h of incubation, as revealed by increasing the proportion of cells in the S phase and the G2/M phase from 29 to 72%. Moreover, exposure to thrombin (> 5 units/ml) for 3 days led to marked clustering of pulp cells. We concluded that thrombin can regulate the growth, cell cycle progression, and functional reorganization of the pulp tissue during pulp healing and inflammatory processes.