Early gene expression analyzed by cDNA microarray and real-time PCR in osteoblasts cultured with chitosan monomer.

Chitosan has a variety of biological activities. Although it has been reported that chitosan promotes osteogenesis in bone lesions, little is known about how it modulates the hard tissue forming cells at the gene level. This study focused on gene expressions in osteoblasts cultured with a super-low concentration of chitosan monomer. cDNA probes were synthesized from isolated RNA and labeled with fluorescent dye. They were hybridized with Human 3.8 II cDNA microarray, and the fluorescent signal was analyzed. cDNA microarray analysis revealed that 10 genes concerning to various signaling-related molecules were expressed at > or =2.0-fold higher signal ratio levels in the experimental group when compared with the control group after 3 days. Real-time PCR analysis showed that chitosan monomer induced an increase in the expression of four signal transduction genes, mitogen-activated protein kinase (MAPK)K3, MAPKKK11, Rac1 and Shc1, together with the alkaline phosphatase gene. These results suggest that a super-low concentration of chitosan monomer could modulate the activity of osteoblastic cells through mRNA levels and that chitosan monomer directly affects signal transduction inside cells.

Chitosan monomer promotes tissue regeneration on dental pulp wounds.

The present study was undertaken to evaluate the applicability of chitosan monomer (D-glucosamine hydrochloride) as a pulp capping medicament. Both in vitro and in vivo experiments were carried out to study the cell metabolism and wound healing mechanisms following the application of chitomonosaccharide. After 3 days of osteoblast culture, alkaline phosphatase (ALP) activity significantly increased in the chitosan group. Reverse transcription polymerase chain reaction analysis revealed that chitosan induced an increase in the expression of ALP mRNA after 3 days and bone morphogenetic protein-2 mRNA after 7 days of osteoblast incubation. Inflammatory cytokine, interleukin (IL)-8, synthesis in fibroblasts was strongly suppressed in the medium supplemented with chitosan monomer. Histopathological effects were evaluated in rat experiments. After 1 day, inflammatory cell infiltrations were observed to be weak when compared with the application of chitosan polymer. After 3 days, a remarkable proliferation of fibroblasts was seen near the applied chitosan monomer. The inflammatory cell infiltration had almost completely disappeared. After 5 days, the fibroblastic proliferation progressed, and some odontoblastic cells appeared at the periphery of the proliferated fibroblasts. These findings indicate that the present study is the first report that chitosan monomer acts as a biocompatibility stable medicament even at the initial stage of wound healing in comparison with the application of chitosan polymer.

Early gene expression analyzed by cDNA microarray and RT-PCR in osteoblasts cultured with water-soluble and low molecular chitooligosaccharide.

Chitosan has a variety of biological activities. However, little is known about how chitosan modulates the hard tissue forming cells. When we cultured an osteoblastic cell line in alpha-MEM supplemented with 10% FBS and 0.005% chitooligosaccharide for 3 days, alkaline phosphatase (ALP) activity was significantly high compared with the control culture group (p<0.05). This study was focused on gene expression in osteoblasts cultured with water-soluble chitooligosaccharide. cDNA probes were synthesized from isolated RNA and labeled with fluorescent dye. They were hybridized with Human 1.0((R)) cDNA microarray, and fluorescent signal was analyzed. cDNA microarray analysis revealed that 16 genes were expressed at >/=1.5-fold higher signal ratio levels in the experimental group compared with the control group after 3 days. RT-PCR analysis showed that chitosan oligomer induced an increase in the expression of two genes, CD56 antigen and tissue-type plasminogen activator. Furthermore, the expression of mRNAs for BMP-2 was almost identical in the experimental and control groups after 3 days of culture, but slightly increased after 7 days of culture with chitosan oligomer. These results suggest that a super-low concentration of chitooligosaccharide could modulate the activity of osteoblastic cells through mRNA levels and that the genes concerning cell proliferation and differentiation can be controlled by water-soluble chitosan.

Surface changes of mineral trioxide aggregate after the application of bleaching agents: electron microscopy and an energy-dispersive X-ray microanalysis.

INTRODUCTION: The aim of this study was to investigate the changes in the surface structure and chemical composition after applying bleaching agents to completely hardened mineral trioxide aggregate. METHODS: A total of 12 samples of MTA blocks were divided into three groups, two different bleaching agents, and a control group. The surface structure was observed using a scanning electron microscope. The changes in elemental composition were analyzed by an energy-dispersive x-ray microanalysis (EDX) system. RESULTS: The surface of the MTA covered with each bleaching agent changed in terms of both color and structure compared with the control. EDX showed that both bleaching agents affected the elemental distribution. A decrease in Ca and an increase in Si were shown, and this tendency was especially pronounced in the higher hydrogen peroxide concentration group. CONCLUSIONS: The acidic conditions induced by bleaching agents brought about the deterioration of the MTA surface. These findings suggest that MTA is an insufficient barrier against tooth bleaching.

Zinc as an essential trace element in the acceleration of matrix vesicles-mediated mineral deposition.

BACKGROUND: Zinc (Zn) has a potent stimulatory effect on osteoblastic bone formation and an inhibitory effect on osteoclastic bone resorption. PURPOSE: The effect of Zn on the function of matrix vesicles (MVs) remains controversial. The purpose of this study was to investigate the effect of Zn on alkaline phosphatase (ALP) activity of osteoblasts and in the initial biological MVs-mediated mineral deposition. STUDY DESIGN: Osteoblasts were treated with varying concentrations of Zn dissolved in culture medium. After three, five, and seven days of culture, ALP activity was assayed. For the detection of a low level of calcium concentration in MVs, X-ray fluorescence (XRF) analyses were applied. The effect of Zn for the transformation of calcium phosphate was analyzed using a scanning electron microscope fitted with an energy dispersive X-ray microanalysis (EDX) system. RESULTS: The ALP activity of osteoblasts in culture medium supplemented with 1 × 10(-5) M of Zn was significantly increased at both five and seven days. XRF data demonstrated higher levels of calcium concentration over time in the Zn-supplemented group. EDX data showed that mineral deposits beginning on day 3 were transformed from whitlockite to calcium phosphate near hydroxyapatite, and that Zn accelerated this transformation. CONCLUSIONS: The proper concentration of Zn increased the ALP activity of osteoblasts after five and seven days of incubation. The present XRF and EDX data suggest that the increase of mineral deposition with Zn exposure for one to five days might be mediated by the activation of ALP and calcium-binding proteins.

Analysis of arsenic in gray and white mineral trioxide aggregates by using atomic absorption spectrometry.

INTRODUCTION: The aim of the present study was to investigate whether the concentration of arsenic (As) released from gray or white mineral trioxide aggregates (MTAs) met the requirement of the International Standards Organization (ISO) for dental cements. METHODS: Sample preparations were carried out according to the ISO methods. After centrifugation of dissolved samples, As (III) concentration in the final supernatant was analyzed by a high-performance atomic absorption spectrophotometer. RESULTS: As (III) concentration from both MTAs was much less than the required value (2 ppm) for dental cements regulated by the ISO. An experiment simulating pulp capping by using MTA revealed that As concentration was also below the standard value of the ISO. The As concentration in white MTA was lower than the value (10 ppb) recommended for tap water and environmental standards. CONCLUSIONS: The present in vitro studies demonstrated that there is no threat to patient health in using commercially available brands of MTA for endodontic practices.

Synchrotron radiation microbeam X-ray fluorescence analysis of zinc concentration in remineralized enamel in situ.

OBJECTIVE: Remineralization is an indispensable phenomenon during the natural healing process of enamel decay. The incorporation of zinc (Zn) into enamel crystal could accelerate this remineralization. The present study was designed to investigate the concentration and distribution of Zn in remineralized enamel after gum chewing. METHODS: The experiment was performed at the Photon Factory. Synchrotron radiation was monochromatized and X-rays were focused into a small beam spot. The X-ray fluorescence (XRF) from the sample was detected with a silicon (Si) (lithium (Li)) detector. X-ray beam energy was tuned to detect Zn. The examined samples were small enamel fragments remineralized after chewing calcium phosphate-containing gum in situ. The incorporation of Zn atom into hydroxyapatite (OHAP), the main component of enamel, was measured using Zn K-edge extended X-ray absorption fine structure (EXAFS) with fluorescence mode at the SPring-8. RESULTS: A high concentration of Zn was detected in a superficial area 10-microm deep of the sectioned enamel after gum chewing. This concentration increased over that in the intact enamel. The atomic distance between Zn and O in the enamel was calculated using the EXAFS data. The analyzed atomic distances between Zn and O in two sections were 0.237 and 0.240 nm. CONCLUSION: The present experiments suggest that Zn is effectively incorporated into remineralized enamel through the physiological processes of mineral deposition in the oral cavity through gum-chewing and that Zn substitution probably occurred at the calcium position in enamel hydroxyapatite.

Early gene expression analyzed by a genome microarray and real-time PCR in osteoblasts cultured with a 4-META/MMA-TBB adhesive resin sealer.

OBJECTIVES: Adhesive resin sealer systems have been applied in endodontics to seal the root canal system. This study was designed to confirm the mechanism of intracellular molecular events in an in vitro cell culture system with a 4-methacryloxyethyl trimellitate anhydride/methylmethacrylate-tri-n-butyl borane (4-META/MMA-TBB) adhesive resin sealer. STUDY DESIGN: The gene expression patterns relating to cell growth and differentiation were examined using a human genome expression microarray and real-time polymerase chain reaction analyses in hard tissue-forming osteoblasts cultured with and without a 4-META/MMA-TBB resin sealer. RESULTS: There was no significant difference in the cell number between the control and adhesive sealer groups. An increased expression of integrin beta, transforming growth factor beta-related protein, craniofacial development protein 1, and PI3K genes was demonstrated. The integrin beta and PI3K genes showed extremely high ratios. CONCLUSIONS: The signal transduction pathway, at least through the PI3K/Akt cascade for cell proliferation and differentiation, can be controlled by some components of this type of adhesive resin sealer.

Gross extrusion of endodontic obturation materials into the maxillary sinus: a case report.

A gross extrusion of endodontic obturation materials occurred from tooth 3 into the right maxillary sinus. The patient had never been conscious of uncomfortable symptoms, both at tooth 3 or buccal regions. A computed tomographic (CT) scan showed cord-like foreign substances extruded from the apex of the tooth and the hyperplasticity of the sinal mucosa. The surgical removal of foreign substances and partial curettage of sinal mucosa were indicated to prevent the possibility of sinus infection. At the 4-month recall, the patient was symptom free. This case emphasizes that an open apex can become potentially dangerous when the vertical condensation method is used. If massive overfilling is recognized radiographically in molar regions, an examination using panoramic radiograph is indispensable to detect the gross extrusion into the maxillary sinus, such as in this case.