Characterization of human dental pulp cells-derived spheroids in serum-free medium: stem cells in the core.

Spheroid models have led to an increased understanding of differentiation, tissue organization and homeostasis. In the present study, we have observed that under a serum-free medium, human dental pulp cells (DPCs) spontaneously formed spheroids, and could survive over 15 weeks. To characterize these spheroids, we investigated their dynamics, microenvironment, cell distribution, molecular profiles, and neuronal/osteogenic potential. Cell tracking assay showed that cells inside the spheroids have very slow cycling. Although the spheroids had hypoxia microenvironments, there were not any massive cell die-offs even after long-term cultivation. Whole mount immunofluorescence staining and histological analysis showed a distribution of stem cells in the central/intermediate zones of spheroids. qRT-PCR analysis demonstrated that the expression of stemness markers NANOG, TP63, and CD44 in the spheroids were much higher than within the monolayer cultures. Gene expression levels of neural markers CDH2, NFM, TUBB3, and CD24 in the spheroids were much higher than the monolayer DPCs and increased in a culture time-dependent manner. Without any neural induction, spheroid-derived cells spontaneously converted into neuron-like cells with positive staining of neural markers HuC/D and P75 under the serum-free medium for about 2 weeks. When the spheroids were transferred into osteogenic medium, they rapidly differentiated into osteo/odontogenic cells, especially the central original cells. Compared to the monolayer DPCs, mineralization in spheroids were significantly increased. This spheroid model offers a study tool to explore the molecular bases of stem cell homeostasis and tissue organization, and can be wildly used for nerve tissue and bone regeneration.

Sensitivity of human dental pulp cells to eighteen chemical agents used for endodontic treatments in dentistry.

To determine the adverse effects against human dental pulp tissue, the sensitivity of human dental pulp cells (D824 cells) to 18 chemical agents used for endodontic treatments in dentistry was examined. The cytotoxicity, as determined by a decrease in colony-forming ability of cells treated with the chemical agents, increased as the concentration increased. As a quantitative measure of the cytotoxic effect, LC(50), the concentration which induces a 50% lethality, was extrapolated from the concentration-response curves. The rank of the chemical agents according to their cytotoxic effect (LC(50)) was sodium arsenite > formaldehyde > hydrogen peroxide > zinc oxide > thymol ≈ iodoform ≈ eugenol > guaiacol > ethylenediaminetetraacetic acid ≈ iodine > procaine > lidocaine ≈ chloramphenicol ≈ m-cresol > calcium hydroxide ≈ sodium hypochlorite ≈ phenol ≈ p-phenolsulfonic acid. To compare the cytotoxicity and the levels of apoptosis and mRNA expression of five genes related to the function of dental pulp tissue, D824 cells treated with the LC(50) concentrations of chemical agents were assayed by the TUNEL method and quantitative reverse transcription polymerase chain reaction analysis, respectively. The inducibility of apoptotic cells and the level of mRNA expression of the genes varied with the chemical agents, indicating that both effects occurred independent of the rank of cytotoxic effect of the chemical agents. The results not only provide information concerning cytotoxicity of various chemical agents to human dental pulp cells, but also show an insight into the diversity of the pharmacodynamic action of the chemical agents.

Three-dimensional epithelial and mesenchymal cell co-cultures form early tooth epithelium invagination-like structures: expression patterns of relevant molecules.

Epithelium invagination is the key feature of early tooth development. In this study, we built a three-dimensional (3D) model to represent epithelium invagination-like structure by tissue engineering. Human normal oral epithelial cells (OECs) and dental pulp stem cells (DPSCs) were co-cultivated for 2-7 weeks on matrigel or collagen gel to form epithelial and mesenchymal tissues. The histological change and gene expression were analyzed by HE staining, immunostaining, and quantitative real-time RT-PCR (qRT-PCR). After 4 weeks of cultivation, OECs-formed epithelium invaginated into DPSCs-derived mesenchyme on both matrigel and collagen gel. OEC-DPSC co-cultures on matrigel showed typical invagination of epithelial cells and condensation of the underlying mesenchymal cells. Epithelial invagination-related molecules, CD44 and E-cadherin, and mesenchymal condensation involved molecules, N-cadherin and Msx1 expressed at a high level in the tissue model, suggesting the epithelial invagination is functional. However, when OECs and DPSCs were co-cultivated on collagen gel; the invaginated epithelium was transformed to several epithelial colonies inside the mesenchyme after long culture period. When DPSCs were co-cultivated with immortalized human OECs NDUSD-1, all of the above-mentioned features were not presented. Immunohistological staining and qRT-PCR analysis showed that p75, BMP2, Shh, Wnt10b, E-cadherin, N-cadherin, Msx1, and Pax9 are involved in initiating epithelium invagination and epithelial-mesenchymal interaction in the 3D OEC-DPSC co-cultures. Our results suggest that co-cultivated OECs and DPSCs on matrigel under certain conditions can build an epithelium invagination-like model. This model might be explored as a potential research tool for epithelial-mesenchymal interaction and tooth regeneration.

Cytotoxic effect of eugenol on the expression of molecular markers related to the osteogenic differentiation of human dental pulp cells.

The cytotoxic effect of eugenol on the expression of molecular markers related to the osteogenic differentiation of human dental pulp cells such as collagen synthesis and the expression of two osteogenesis-related genes, alkaline phosphatase (ALP) and bone sialoprotein (BSP), was studied using human dental pulp cells (D824 cells). Cellular growth and survival were decreased by treatment of cells with eugenol in a concentration-dependent manner. The incorporation rate of [(3)H] proline into the acid-insoluble fraction and the synthesis of type I-V collagens were also reduced by treatment of cells with eugenol in a concentration-dependent fashion. The mRNA expression of ALP was scarcely affected in cells exposed to eugenol, whereas the mRNA and protein expression of BSP was down-regulated depending on the concentrations of eugenol. The results suggest that because collagen synthesis and BSP expression play a critical role in hard tissue formation, eugenol used for endodontic treatment may give rise to cytotoxic effects to the normal function of stem cells reported to exist in human dental pulp tissue and periodontal ligament.

Induction of mRNA expression of osteogenesis-related genes by guaiacol in human dental pulp cells.

To investigate the stimulating effect of endodontic medications on the mRNA expression of some osteogenesis-related genes associated with reparative dentinogenesis and hard-tissue formation, human dental pulp cells (D824 cells) were treated with calcium hydroxide (Ca (OH)(2)), formocresol, or guaiacol. The effect on growth was determined by growth curves of D824 cells treated for 1-3 days with 0.03-0.3 mM Ca (OH)(2), 0.0007%-0.0014% formocresol, or 0.24-2.43 mM guaiacol. The mitotic activity of individual cells and the mRNA expression of the osteogenesis-related genes for alkaline phosphatase (ALP), type I collagen (COL-1), and bone sialoprotein (BSP) in the cells treated for 24 h with the same concentrations of the medications as described above were determined by colony-forming efficiency and by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis, respectively. Cellular growth and mitotic activity were scarcely affected by Ca (OH)(2), but were significantly reduced by formocresol or guaiacol. The mRNA expression of the osteogenesis-related genes was little affected by Ca (OH)(2) or formocresol, but was significantly enhanced by guaiacol. The results indicate that guaiacol may stimulate the mRNA expression of genes associated with reparative dentinogenesis and hard-tissue formation in human dental pulp cells, suggesting that the novel property of guaiacol provides new insights into the utilization of guaiacol in endodontic therapy.

Clastogenic activity of seven endodontic medications used in dental practice in human dental pulp cells.

Numerous and varied chemical agents are used for endodontic treatments in dental practice. Endodontic medications are administered directly to the teeth in relatively high concentrations and chemical agents applied to enamel or dentin can penetrate the dental pulp tissue and circulate through the body in the bloodstream. In the present study, to assess safety regarding mutagenicity, we investigated the ability of seven endodontic medications to induce chromosome aberrations in human dental pulp cells. Chromosome aberrations were induced in cells treated with each of six endodontic medications, eugenol, guaiacol, modified phenol, phenol, thymol, and zinc oxide. The other endodontic medication, zinc chloride, failed to induce chromosome aberrations in the presence or absence of exogenous metabolic activation. The percentages of cells with polyploid or endoreduplication were not enhanced by any of the endodontic medications tested. Our results indicate that the endodontic medications that exhibited a positive response are potentially mutagenic to human cells.

Ability of root canal antiseptics used in dental practice to induce chromosome aberrations in human dental pulp cells.

Root canal antiseptics are topically applied to root canals within the pulpless teeth to treat the root canal and periapical infections. Because the antiseptics that are applied to root canals can penetrate through dentin or leak out through an apical foramen into the periodontium and distribute by the systemic circulation, it is important to study the safety of these antiseptics. In the present study, we examined the ability to induce chromosome aberrations in human dental pulp cells of five root canal antiseptics, namely, carbol camphor (CC), camphorated p-monochlorophenol (CMCP), formocresol (FC), calcium hydroxide, and iodoform which are most commonly used in dental practice. Statistically significant increases in the levels of chromosome aberrations were induced by CC, FC, or iodoform in a concentration-dependent manner. Conversely, CMCP and calcium hydroxide failed to induce chromosome aberrations in the absence or presence of exogenous metabolic activation. The percentages of cells with polyploid or endoreduplication were enhanced by FC or iodoform. Our results indicate that the root canal antiseptics that exhibited a positive response are potentially genotoxic to human cells.

Assessment of genotoxicity of 14 chemical agents used in dental practice: ability to induce chromosome aberrations in Syrian hamster embryo cells.

To assess the genotoxicity of 14 chemical agents used as locally applied agents in dental practice, the ability of these agents to elicit chromosome aberrations was examined using Syrian hamster embryo (SHE) cells. Chromosome aberrations in SHE cells were induced by treatment with three of eight chemical agents used as endodontic medicaments, i.e. ethylenediaminetetraacetic acid (EDTA), formocresol (a mixture of formalin and tricresol), and sodium arsenite. The other five chemical agents, i.e. chloramphenicol, p-chlorophenol, p-phenolsulfonic acid, sodium hypochlorite, and tetracycline hydrochloride exhibited a negative response for chromosome aberrations. Assessment of three dyes used for disclosing dental plaque showed chromosome aberrations induced by basic fuchsin but not by acid fuchsin and erythrosine B. Three local anesthetics, lidocaine hydrochloride, prilocaine hydrochloride, and procaine hydrochloride, were negative for chromosome aberrations. Among the ten chemical agents that exhibited a negative response in the assay, p-chlorophenol, sodium hypochlorite, and erythrosine B induced chromosome aberrations in SHE cells when treated in the presence of exogenous metabolic activation. The percentages of cells with polyploidy or endoreduplication were enhanced by formocresol, sodium arsenite, p-chlorophenol, p-phenolsulfonic acid, sodium hypochlorite, erythrosine B, prilocaine hydrochloride, and procaine hydrochloride in the absence or presence of exogenous metabolic activation. Our results indicate that the chemical agents that had a positive response in the present study are potentially genotoxic to mammalian cells.

Human dental mesenchymal stem cells and neural regeneration.

Nerve tissue presents inherent difficulties for its effective regeneration. Stem cell transplantation is considered an auspicious treatment for neuronal injuries. Recently, human dental mesenchymal stem cells (DMSCs) have received extensive attention in the field of regenerative medicine due to their accessibility and multipotency. Since their origin is within the neural crest, they can be differentiated into neural crest-derived cells including neuron and glia cells both in vitro and in vivo. DMSCs are also able to secrete a wide variety of neurotrophins and chemokines, which promote neuronal cells to survival and differentiation. Experimental evidence has shown that human DMSCs engraftment recovered neuronal tissue damage in animal models of central nervous system injuries. Human DMSCs can be a new hope for treatment of nervous system diseases and deficits such as spinal cord injury, stroke and Parkinson's disease.

Expression status of mRNA for sex hormone receptors in human dental pulp cells and the response to sex hormones in the cells.

OBJECTIVES: Sex hormone receptors are reported to be present in human dental pulp (HDP) cells. The purpose of this study was to examine the biological significance of oestrogen and androgen receptors (ER and AR, respectively) in HDP cells. DESIGN: We isolated HDP cells expressing ER- and AR-mRNAs and investigated the expression status of the receptors and the response to sex hormones in the cells. RESULTS: HDP cells expressing ER- and/or AR-mRNAs had the ability to form alizarin red S-positive nodules in which calcium and phosphorus were deposited in vitro and to differentiate into odontoblasts-like cells and dentine-like tissue in vivo. Individual clones isolated from HDP cells exhibited a different expression pattern of mRNA for ER and AR. Some clones expressed ERα- and/or ERß-mRNAs and the others coexpressed ER- and AR-mRNAs. Using the Ingenuity software, we found that 17ß-estradiol (E2) and dihydrotestosterone (DHT) could act directly on HDP cells through ER-or androgen signalling-mediated mechanisms. E2 or DHT stimulated the mRNA expression for genes related to odontogenesis of dentine-containing teeth and odontoblast differentiation, suggesting that ER and AR in HDP cells may be involved in dentinogenesis. CONCLUSIONS: Our findings provide new insights into the biological significance of sex hormone receptors in HDP cells.

Immortalization of normal human gingival keratinocytes and cytological and cytogenetic characterization of the cells.

Most in vitro studies of oral carcinogenesis in human cells are carried out with oral keratinocytes immortalized by human papillomavirus type 16 DNA. However, because various etiological factors for oral cancer are known, it is important to establish new human keratinocyte cell lines useful for studying the mechanism of oral carcinogenesis. Normal human gingival keratinocytes in secondary cultures grown in serum-free medium were either transfected with origin (-) SV40 DNA or sequentially transfected with origin (-) SV40 DNA and human c-fos. The transfected cells were continually passaged and analyzed for cytological and cytogenetic characterizations. Four immortal cell lines were grown for over 1100 days in culture and maintained a vigorous growth for over 250 population doublings. They expressed SV40 T antigen, cytokeratins 8 and 18, and E-cadherin, and overexpressed the c-Fos protein. The immortal cell lines had telomerase activity but lacked transformed phenotypes on soft agar or in nude mice. Each cell line had nonrandom chromosomal abnormalities and minisatellite alterations. One of the immortal cell lines, NDUSD-1, retained the capability to deposit calcium, which was also demonstrated in normal human gingival keratinocytes by alizarin red staining, indicating the possibility that NDUSD-1 cells may retain some natural characteristics of normal gingival keratinocytes. Because the oral ectoderm plays an important role in tooth development, these immortal cell lines may be useful in various experimental models for investigations of oral biology and oral carcinogenesis.

Assessment using human dental pulp cells of clastogenicity of antiseptics used in dental practice and agents for root canal enlargement and cleaning.

Numerous and varied chemical agents are used as topically applied drugs in dental practice. As they are administered directly to the oral cavity, it is important to study the safety of these agents. In the present study, to assess safety regarding mutagenicity, we investigated the abilities of six antiseptics to induce chromosome aberrations in human dental pulp cells. The antiseptics tested were benzalkonium chloride, benzethonium chloride, iodine glycerin, iodine tincture, oxydol, and povidone-iodine. In addition, we tested two agents used for root canal enlargement and cleaning, ethylenediaminetetraacetic acid and sodium hypochlorite. Chromosome aberrations were induced only in cells treated with the highest concentration of iodine tincture for 30 h. The other chemical agents failed to induce chromosome aberrations in the presence or absence of exogenous metabolic activation. The concentration of iodine tincture to which patients are exposed in dental practice is 1000-fold the concentration that induced chromosome aberrations in the present study. Our findings suggest that iodine tincture is mutagenic to human cells.

Effects of minocycline and doxycycline on cell survival and gene expression in human gingival and periodontal ligament cells.

OBJECTIVES AND BACKGROUND: Periodontitis is an infectious disease in the gingival crevice caused by periodontopathic bacteria, including Porphyromonas gingivalis, Prevotella intermedia, Actinobacillus actinomycetemcomitans, and Tennerella forsythensis, and antibacterial agents are directly administered to the site of infection to treat it. To maximize the therapeutic effects while reducing the adverse effects, the antibacterial agents should be administered at concentrations greater than their MIC(90) doses required to inhibit the growth of 90% of periodontopathic bacteria and the administration should not damage the periodontal tissue. One approach for estimating cellular damage in the periodontal tissue caused by the administration is to assay cytological damages following exposures of cultured human cells derived from periodontal tissues to antibacterial agents. In the present study, we investigated the cytotoxic effect of minocycline (MINO) and doxycycline (DOX) by using a human gingival fibroblast cell line, a human gingival epithelial cell line, and a human periodontal ligament fibroblast cell line. We also used these cell lines to study the effect of MINO or DOX on the mRNA and protein expressions of genes associated with the differentiation of fibroblasts and the proliferation, differentiation, or cellular adhesion important to the epithelial regeneration of the periodontal attachment. METHODS: The cytotoxic effect of MINO or DOX was measured as a decrease in cell survivals. The effects of these antibiotics on the mRNA and protein expressions in the cell lines were studied by reverse transcription-polymerase chain reaction (RT-PCR) and western blot analyses, respectively. RESULTS: The maximum concentration of MINO or DOX that has little effect on the cell survivals and the mRNA and protein expressions of genes for alkaline phosphatase, type I procollagen, keratinocyte growth factor receptor, keratin 18 or 8/18, integrin beta1, integrin beta4, and laminin 5gamma2 was 10 or 30 microm, respectively, which are greater than their MIC(90) doses against periodontopathic bacteria described above. CONCLUSIONS: These findings suggest that little, if any, cellular damage would be expected with topical administration of MINO or DOX to the periodontal pocket at a dose equivalent to the MIC(90). It is important to note, however, that the extrapolation of these findings to in vivo conditions has yet to be undertaken.

In vitro chromosome aberration tests using human dental pulp cells to detect the carcinogenic potential of chemical agents.

To examine if human dental pulp cells are useful for assessing the carcinogenic potential of chemical agents, we cultured human dental pulp cells from adults and studied the ability of chemical agents known to be carcinogenic to induce chromosome aberrations in these cells. We confirmed that human dental pulp cells in primary or secondary cultures had the capability of accumulating calcium in vitro as detected by Alizarin red staining and generating dentin-like tissue in immunocompromised mice. These phenotypes were maintained even in cells at seven passages. Next, we examined if chromosome aberrations were induced by exposure of human dental pulp cells (designated here as D824 cells) at seven to nine passages to chemical agents with carcinogenic activity. Statistically significant increases in the frequencies of chromosome aberrations were induced in D824 cells treated with a direct-acting carcinogen, mitomycin C, for 3 h. Chromosome aberrations were also induced at statistically significant levels in D824 cells treated with an indirect-acting carcinogen, cyclophosphamide, for 2 h in the presence of exogenous metabolic activation with rat liver postmitochondrial supernatant. Cyclophosphamide failed to induce chromosome aberrations in the absence of exogenous metabolic activation. Although the reliability of chromosome aberration tests using human dental pulp cells remains to be validated by studying the ability of various other chemical agents with or without carcinogenic activity to induce chromosome aberrations, this chromosome aberration test system may be useful for carcinogenic risk assessment in the target cells.

Ability of 13 chemical agents used in dental practice to induce sister-chromatid exchanges in Syrian hamster embryo cells.

To evaluate the genotoxic potential of 13 chemical agents used in dental practice, the abilities of these agents to induce sister-chromatid exchanges (SCEs) were examined using Syrian hamster embryo (SHE) cells. Statistically significant increases in the frequencies of SCEs were observed in SHE cells treated with all seven of the chemical agents used as endodontic medicaments: p-chlorophenol, m-cresol, formaldehyde, guaiacol, hydrogen peroxide, p-phenolsulfonic acid, and sodium hypochlorite (P < 0.01; Student t test). Assessment of two chemical agents that are applied to the oral mucosa as antiseptics showed that SCEs were induced by iodine (P < 0.01), but not by chlorhexidine. Of three chemical agents that are used as dyes for disclosing dental plaque, erythrosine B had no effect on SCE induction, while acid fuchsin and basic fuchsin increased the SCE frequencies in SHE cells (P < 0.01). Glutaraldehyde, which is used as a disinfectant for dental instruments and impressions, also induced SCEs (P < 0.01). Because SCE assays are used as a sensitive indicator for evaluating genetic toxicity of chemicals, the chemical agents that had a positive response in the present study are potentially genotoxic to mammalian cells.

Ability of fourteen chemical agents used in dental practice to induce chromosome aberrations in Syrian hamster embryo cells.

To assess the genotoxicity of 14 chemical agents used in dental practice, the ability of these agents to induce chromosome aberrations was examined using Syrian hamster embryo (SHE) cells. Statistically significant increases in the frequencies of chromosome aberrations were induced in SHE cells treated with 7 of 10 chemical agents used as endodontic medicaments, that is, carbol camphor, m-cresol, eugenol, guaiacol, zinc oxide, hydrogen peroxide, and formaldehyde. The other 3 chemical agents, that is, thymol, glutaraldehyde, and iodoform, did not increase the levels of chromosome aberrations. Of the 4 chemical agents that are used as an antiseptic on the oral mucosa, chromosome aberrations were induced by iodine, but not by the other 3 antiseptics, benzalkonium chloride, benzethonium chloride, and chlorhexidine. Among the 6 chemical agents exhibiting a negative response in the assay, only thymol induced chromosome aberrations in the presence of exogenous metabolic activation. Our results indicate that chemical agents having a positive response in the present study are potentially genotoxic to mammalian cells and need to be studied further in detail.

Cell-transforming activity of fourteen chemical agents used in dental practice in Syrian hamster embryo cells.

Fourteen chemical agents used in dental practice were assessed for their cell-transforming activity using the Syrian hamster embryo (SHE) cell transformation assay system. The cell-transforming activity was quantitatively assessed by the frequency of morphological transformation (MT) in SHE cells induced by these agents. MT was induced by m-cresol, guaiacol, formaldehyde, sodium hypochlorite, hydrogen peroxide, sodium arsenite, acid fuchsin, and basic fuchsin, but not by p-chlorophenol, p-phenolsulfonic acid, glutaraldehyde, and erythrosine B. Iodine and chlorhexidine exhibited positive and pseudopositive responses, respectively. The chemical agents exhibiting a negative or pseudopositive response neither induced nor enhanced MT even in the presence of exogenous metabolic activation.

Quantitative comparison of the cytocidal effect of seven macrolide antibiotics on human periodontal ligament fibroblasts.

The cytocidal effect of seven macrolide antibiotics on human periodontal ligament fibroblasts (Pel cells) was studied. Pel cells were exposed for 48 h to erythromycin (EM), clarithromycin (CAM), roxithromycin (RXM), azithromycin (AZM), josamycin (JM), midecamycin (MDM), and rokitamycin (RKM), and allowed to form colonies. The cytocidal effect of the macrolides was measured as a decrease in colony-forming efficiency and was found to increase with the concentration. To obtain a quantitative measure of the cytocidal effect, the LD50, i.e. the concentration that decreases colony-forming efficiency 50% relative to control cells, was extrapolated from the concentration-response curves. The rank of the macrolides according to their cytocidal effect (LD50) was RKM > RXM > CAM > AZM > JM > MDM approximately EM. RKM, RXM, CAM, AZM, and JM were at least 1.7-12.2 times more cytocidal than MDM or EM. When extrapolated from the concentration-response curves, the relative survival of the Pel cells exposed to each of the macrolides at the MIC90 concentrations for periodontopathic bacteria was estimated to be: > or = 53.8% for RKM, > or = 92.7% for RXM, > or = 94.6% for CAM, > or = 97.1% for AZM, and > or = 86.2% for EM. The effect of the antibiotics on the mRNA expression of alkaline phosphatase (ALP) and type I procollagen (COL) was examined in Pel cells exposed for 48 h to RXM, CAM, AZM, and EM, which exhibited strong, moderate, and weak cytocidal activity. The constitutive levels of both ALP and COL mRNA were retained in cells exposed to RXM at < or = 3 microM, CAM at < or = 10 microM, and AZM or EM at < or = 3 microM. The MIC90 against periodontopathic bacteria is < or = 4.8 microM for RXM, 5.3 microM for CAM, 2.7 microM for AZM, and 21.8 microM for EM. These results suggest that topical administration of CAM or AZM to the gingival crevice at their MIC90 concentration for periodontopathic bacteria would have little adverse effect on the growth and differentiation of the periodontal ligament. It is important to note, however, that these findings have yet to be extrapolated to in vivo conditions.

Effects of eight antibacterial agents on cell survival and expression of epithelial-cell- or cell-adhesion-related genes in human gingival epithelial cells.

OBJECTIVE AND BACKGROUND: Our previous studies suggest that little adverse effect on the growth of the periodontal ligament would be expected, if tetracycline, minocycline, ofloxacin, roxithromycin, clarithromycin, and azithromycin were topically administered to the periodontal pocket at their MIC90 doses required to inhibit the growth of 90% of periodontopathic bacteria, including Porphyromonas gingivalis, Prevotella intermedia, and Actinobacillus actinomycetemcomitans. In the present study, we investigated the cytocidal effects of eight antibacterial agents on the human gingival epithelial cell line NDUSD-1. We also used NDUSD-1 cells to examine the effects of these agents on the mRNA and protein expressions of genes associated with the proliferation, differentiation, or cellular adhesion important to the epithelial regeneration of the periodontal attachment. METHODS: The cytocidal effect of the test agents was measured as a decrease in cell survival. To obtain a quantitative measure of the cytocidal effect, the LD50, i.e. the concentration which results in a 50% decrease in cell survival relative to the controls, was extrapolated from the concentration-response curves. The effects of the agents on the mRNA and protein expressions in NDUSD-1 cells were studied by reverse transcription-polymerase chain reaction (RT-PCR) and western blot analyses, respectively. RESULTS: The cytocidal effect increased in a concentration-dependent manner as the concentration of each of the eight test agents increased. The order of the agents according to their cytocidal effects (LD50) was minocycline > tetracycline > enoxacin > clarithromycin > roxythromycin approximately ofloxacin > azithromycin > erythromycin. The cytocidal effects of minocycline, tetracycline, enoxacin, clarithromycin, roxythromycin, ofloxacin, and azithromycin ranged from 1.2 to 23.2 times greater than that of erythromycin. The maximum non-cytocidal concentrations (MNCCs) of these agents for NDUSD-1 cells were: 0.3 microm for minocycline, 1 microm for tetracycline, 3 microm for ofloxacin and erythromycin, 10 microm for enoxacin, clarithromycin, and azithromycin, and 100 microm for roxythromycin. The MNCCs of ofloxacin, azithromycin, clarithromycin, and roxythromycin were greater than their MIC90 concentrations for periodontopathic bacteria described above. The effects on the mRNA and protein expressions of epithelial-cell- or cell-adhesion-related genes were examined in NDUSD-1 cells exposed to clarithromycin, roxythromycin, ofloxacin, and azithromycin at their MNCCs. None of the agents affected the mRNA expressions of five genes: keratinocyte growth factor receptor, keratin 18, integrin beta1, integrin beta4, and laminin 5gamma2. Clarithromycin and ofloxacin slightly decreased the protein expression of integrin beta4. Roxythromycin markedly decreased the protein expressions of integrin beta4 and laminin 5gamma2. Azithromycin had little inhibitory effects on the protein expressions of any of the five genes. CONCLUSIONS: These results suggest that little, if any, adverse effects on growth, differentiation, and adhesion of basal epithelial cells would be expected with topical administration of clarithromycin, ofloxacin or azithromycin to the periodontal pocket at a dose equivalent to the MIC90. It is important to note, however, that the extrapolation of these findings to in vivo conditions has yet to be undertaken.