Application Of CLSM In Researching On The Concentration Of Ca²⁺ In Dental Pulp Cells And Microleakage Of Dental Filling Materials

Confocal Laser Scanning Microscopy(CLSM)is one of the widest used and the highest resolution image experiment instruments in biology.The excellent characteristics of CLSM make it possible to be employed gradually in stomatology research.Based on the dental pulp cell biology and marginal sealing of artificial filling materials,this study focused on the application of CLSM in researching changes of calcium ion(Ca²⁺)concentration in dental pulp cells and microleakage of dental filling materials,further,to enrich the basic theory in tooth and dental pulp research and to provide guidance for clinical practice.Objectives:Using CLSM:(1)to examine the effect of hydroxyapatite(HA) on intracellular free calcium ion(Ca²⁺)of in vitro cultured human dental pulp cells and determine whether HA is taking action in the process of Ca²⁺adjustment in the pulp cells;(2)to observe microleakage conditions of three commonly used dental materials——amalgam,composite resin and glass ionomer after being filled in isolated human teeth,and to compare their sealing ability for selection of materials in clinical use;(3)to evaluate the microleakage of dental filling materials in isolated human teeth observed by CLSM and metallographic microscope in order to provide the better means for further research.Methods:CLSM was used to detect the changes of intracellular calcium ion concentration([Ca²⁺]i)in the cultured human dental pulp cells in vitro after stimulation by HA.Extracted human caries-free teeth with occlusal cavity preparation were randomly divided into 3 groups and filled with amalgam,composite resin and glass ionomer respectively,CLSM was used to evaluate quantitively the microleakage on the interfaces of the teeth.Glass ionomer cement was optimized as the filling material for the experimental cavities,CLSM and metallographic microscope were used for measuring the microleakage depths on the marginal interfaces.Results:After HA stimulation,the[Ca²⁺]i increased quickly in all groups no matter how long the co-incubation time was.The fluorescence intensity increased for instantaneous[Ca²⁺]i was 60%in the control group,13%in the calcium hydroxide [Ca(OH)₂]group,and 8.7%in HA group.The fluorescent dye penetrations were observed in all three groups with different degree,the microleakage depth of glass-ionomer cement group was 23.63±3.06μm,silver amalgam group was 31.68±10.97μm and composite resin group was 32.86±11.67μm.The difference of microleakage between glass-ionomer cement and composite resin group was significant.In the subgroups,using glass-ionomer cement for pretreatment caused less microleakage than filling silver amalgam directly.Complete etch primer system was better statistically than self-etching in reducing microleakage.Both CLSM and metallographic microscope can detect dye penetration in the margin of the interfaces, but when filled with the traditional glass ionomer cement and resin modified glass ionomer cement,the microleakage depths observed by CLSM were 22.57±2.59μm and 21.93±1.51μm,while the values measured by metallographic microscope were 33.18±5.59μm and 32.30±3.76μm.The results showed statistical significance(P ＜0.05).Conclusions:HA increased[Ca²⁺]i in cultured human dental pulp cells in vitro,therefore it is considered that HA participates in the process of Ca²⁺ transportation in dental pulp cells.For dental filling materials,glass-ionomer cement resulted in less marginal microleakage and using glass ionomer cement for pretreatment can reduce microleakage significantly before silver amalgam filling. Complete etching composite resin is better than self-etching in reducing marginal microleakage.The CLSM is a preferred experimental instrument in microleakage evaluation for its simplicity in specimen preparation and quantitative detection as well as high specificity in microleakage observation of dental filling materials.