Shigeru Kobayashi

Extracts of Actinobacillus actinomycetemcomitans Induce Apoptotic Cell Death in Human Osteoblastic MG63 Cells

Whether an extracellular component of periodontal-disease-causing bacteria induces apoptotic cell death in bone-related cells is unknown. To study the effects on osteoblasts of extracts obtained from sonicated Actinobacillus actinomycetemcomitans and Prevotella intermedia, we cultured human osteoblastic cell lines MG63 and Saos-2 cells and mouse osteoblastic cell line MC3T3-E1 cells in the presence of such extracts. The addition of the extracts from Actinobacillus actinomycetemcomitans induced cell death in MG63 cells in a dose- and time-dependent fashion over the concentration range of 0.1 to 10 μg/mL. By contrast, the extracts from Prevotella intermedia did not induce cell death in these cells, even in the presence of 10 μg/mL protein. By using the Hoechst 33342 staining technique, we observed marked nuclear condensation and fragmentation of chromatin in MG63 cells treated with the extracts of Actinobacillus actinomycetemcomitans. DNA ladder formation, a hallmark of apoptosis, also was detected in MG63 cells treated with extracts from Actinobacillus actinomycetemcomitans. In MG63 cells, DNA ladder formation was dose-dependent, with a maximal effect at a concentration of 10 μg/mL, and time-dependent, from 12 to 48 hrs. However, the extracts from Prevotella intermedia did not induce DNA fragmentation in MG63, Saos-2, or MC3T3-E1 cells. The extracts from Actinobacillus actinomycetemcomitans did not induce cell death and DNA fragmentation in Saos-2 and MC3T3-E1 cells. Sonicated extracts of Actinobacillus actinomycetemcomitans that had been treated with heat and trypsin did not induce DNA ladder formation in MG63 cells, suggesting that the apoptosis-inducing factors are proteinaceous. Cycloheximide prevented the Actinobacillus actinomycetemcomitans-induced DNA ladder formation in MG63 cells in a dose-dependent fashion, suggesting that new gene transcription and protein synthesis are regulated for Actinobacillus actinomycetemcomitans-induced apoptosis in MG63 cells. Our results indicate that apoptosis in alveolar bone cells induced by Actinobacillus actinomycetemcomitans plays an important role in periodontal diseases.

Prilocaine induces apoptosis in osteoblastic cells.

PurposeTo determine whether prilocaine, a local anesthetic, induces apoptosis in osteoblastic cells.MethodsAfter reaching subconfluence, human osteoblastic Saos-2 and MG63 cells and mouse osteoblastic MC3T3-E1 cells were exposed for 48 hr to varying concentrations of prilocaine up to 10 mM and the cytotoxicity of the cells was analyzed by phase-contrast microscopy and WST-1 assay. Saos-2 cells treated for 48 hr with 5 mM prilocaine were stained with Hoechst 33342 and nuclear fragmentation was examined under a fluorescence microscope. DNA was extracted from the cells treated with 5 mM prilocaine and DNA ladder formation (a hallmark of apoptosis) was analyzed by agarose gel electrophoresis.ResultPrilocaine induced cell death in Saos-2 cells in a dose- and time-dependent manner up to the concentration of 10 mM. Marked nuclear condensation and fragmentation of chromatin were observed in the prilocainetreated cells. DNA ladder formation also was induced by prilocaine treatment. Prilocaine-induced DNA ladder formation was dose-dependent with maximal effect at a concentration of 5 mM and was time-dependent from 12 to 48 hr. DNA ladder formation was also induced by prilocaine treatment in human osteoblastic MG63 cells and mouse osteoblastic MC3T3-E1 cells. Cycloheximide prevented prilocaine-induced apoptosis in Saos-2 cells in a dose-dependent fashion up to 20 μM as determined by WST-1 assay and DNA ladder formation in agarose gel electrophoresis.ConclusionOsteoblastic cells treated with prilocaine exhibit both morphological and biochemical features indicative of apoptosis. The apoptotic mechanisms involve transcriptional regulation of specific proteins or protein synthesis.RésuméObjectifDéterminer si la prilocaïne, un anesthésique local, a induit l’apoptose de cellules ostéoblastiques.MéthodeAprès avoir atteint le stade de sous-confluence, des cellules ostéoblastiques humaines Saos-2 et MG63 et ostéoblastiques de souris MC3T3-E1 ont été exposées pendant 48 h à des concentrations variables de prilocaïne allant jusqu’à 10 mM et la cytotoxicité des cellules a été analysée par microscopie en contraste de phase et par dosage WST-1. Les cellules Saos-2 traitées pendant 48 h avec 5 mM de prilocaïne ont été colorées avec le Hoechst 33342 et la fragmentation nucléaire a été examinée sous microscopie en fluorescence. L’ADN a été extrait et la formation d’échelle d’ADN (signe cardinal de l’apoptose) a été analysée par électrophorèse sur gel d’agarose.RésultatsLa prilocaïne a induit la mort des cellules Saos-2 d’une manière dépendante de la dose et du temps jusqu’à une concentration de 10 mM. En effet, la prilocaïne a induit dans les cellules traitées une condensation nucléaire marquée et la fragmentation de chromatine de même que la formation d’échelle d’ADN. La formation d’ADN, dépendante de la dose, a connu son effet maximal avec une concentration de 5 mM et était dépendante du temps entre 12 et 48 h. La formation d’ADN a été aussi induite par la prilocaïne dans les cellules humaines MG63 et dans les cellules de souris MC3T3-E1. La cycloheximide a empêché l’apoptose induite par la prilocaïne de se produire dans les cellules Saos-2 d’une manière dépendante de la dose pour une concentration jusqu’à 20 μM comme l’ont déterminé le dosage WST-1 et la formation d’échelle d’ADN dans électrophorèse sur gel d’agarose.ConclusionLes cellules ostéoblastiques traitées avec de la prilocaïne ont présenté des caractéristiques morphologiques et biochimiques indicatrices d’apoptose. Les mécanismes de l’apoptose impliquent la régulation de la transcription de protéines spécifiques ou de synthèse protéique.

Inhibitors of protein synthesis and RNA synthesis protect against okadaic acid-induced apoptosis in human osteosarcoma cell line MG63 cells but not in Saos-2 cells

Abstract: In a previous study, we demonstrated that the protein phosphatase inhibitors, okadaic acid and calyculin A, induced apoptosis in human osteosarcoma cell lines, Saos-2 and MG63 cells. In the present study, to determine if new gene transcription and protein synthesis are required for okadaic acid-induced apoptosis in Saos-2 and MG63 cells, the cells were treated for 48 h with varying concentrations of the inhibitors of protein or RNA synthesis, i.e., cycloheximide, actinomycin D, and puromycin, in the presence of a fixed dose of okadaic acid. All these reagents in different concentrations prevented the okadaic acid-induced apoptosis in MG63 cells in a dose-dependent fashion. The same concentrations of cycloheximide, actinomycin D, or puromycin alone did not induce any apoptotic features in MG63 cells. However, not all the aforementioned reagents affected okadaic acid-induced apoptosis in Saos-2 cells. Okadaic acid-induced and cycloheximide-prevented apoptosis was shown by phase-contrast microscopy, WST-1 assay, direct visualization of nuclear condensation and fragmentation of chromatin, and the characteristic DNA ladder formation on agarose gel electrophoresis. The present results indicate that the induction of new cell death genes and ongoing protein synthesis may have a role in okadaic acid-induced apoptosis in MG63 cells and that such proteins are not required in Saos-2 cells.

The Healing Process of Tooth Extraction Wounds Observed by Scanning Electron Microscopy

Morphological studies on the reparative process of the extraction wound have been carried out by many investigators, but the cell level scanning electron microscopic morphological investigation remains unclarified. In the present study, the healing processes of tooth extraction wounds in rats were observed histologically, roentgenologically and scanning electron microscopically. The results obtained were as follows: 1. The extraction wound healing process is divided into four stages in general: blood clot stage, granulation stage, bone trabeculae stage and bone reconstruction stage. But there were no clear borderlines between these stage; in an extraction socket plural healing stages could be observed concurrently. 2. Prevention of infection in extraction wound was as follows: a number of inflammatory cells, especially polymorphonucrear leucocytes (PMNL), diffused in the blood clot in early stage and the PMNL then gradually localized in the upper part of the blood clot. When the gingival epithelium filled the edentulous area, almost all of