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Root-end Filling Materials Alter Fibroblast Differentiation

¹ Department of Endodontics, Center of Excellence in Oral and Craniofacial Biology, Louisiana State University Health Science Center, School of Dentistry; and
² Department of Cell Biology and Anatomy, Center of Excellence in Oral and Craniofacial Biology, Box 128, Louisiana State University Health Science Center, School of Dentistry, 1100 Florida Avenue, New Orleans, LA 70119;

View larger version (30K): [in a new window]   Figure 1. Survival and proliferation of fibroblasts on root-end filling materials. The survival and proliferation of gingival and PDL fibroblasts grown (A) on tissue culture plastic were examined fluorometrically as a measure of total DNA content within these cells. The effects of several root-end filling materials (B, Bone; C, MTA; D, Amalgam; E, HICR; F, ZOEC) were also examined. Gingival fibroblasts (triangles) and PDL fibroblasts (squares) were compared for freshly prepared materials (solid lines; solid symbols) and those thoroughly washed for 2 wks (dashed lines; open symbols). The error bars represent the standard deviation of 4 samples for each material. Plastic represents the results of 2 independent experiments, neither involving washing. ANOVA analysis was applied to all the values, and statistical significance was determined at p < 0.01. Data points represent a typical experiment that was repeated in triplicate.

View larger version (63K): [in a new window]   Figure 2. Transcript alteration by root-end filling materials. Semi-quantitative analysis of transcript expression by periodontal ligament and gingival fibroblasts was performed with RT-PCR. The expression of transcripts associated with hard-tissue formation was examined when these fibroblasts were cultured with different root-end filling materials. ß1 integrin is an ECM protein found in all cell types and was used as a standard control for template cDNA quality and quantity. Alk Phos = alkaline phosphatase; BSP = Bone sialoprotein; MTA = mineral trioxide aggregate.

View larger version (28K): [in a new window]   Figure 3. Changes in relative transcript expression. The relative expressions of specific transcripts were compared for cells (either gingival fibroblasts [GF] or periodontal ligament fibroblasts [PDLF]) treated with root-end filling materials (HICR and MTA) with respect to control cultures grown on plastic. All cells were treated with 10 mM dexamethasone to stimulate differentiation to a mineralized phenotype. The RNA levels in all samples were first normalized to depict uniform expression of the S15 ribosomal subunit. The Fig. depicts the relative expression of differentially expressed transcripts as the mean ± standard deviation of 3 independent experiments. The relative levels of expression for a specific transcript are expressed as a ratio of the expression by cells grown in the presence of the material and those grown on plastic, where a value of 1 indicates identical levels of expression. The dashed rectangle represents the area in which relative expression values are not statistically different from one another. In this study, we determined statistically that expression levels were significantly different if there was a five-fold or greater difference in expression, based upon the average variance between samples with a group (40%), the number of independent samples evaluated (4), and a confidence level of greater than 99% by ANOVA analysis. Each point represents the mean of 3 samples. Error bars represent standard deviation from the mean. Ob Spec = osteoblast specific factor-1, Ob Cys = osteoblast cysteine-rich protein, Ob Stim = osteoblast-stimulating factor, Alk Phos = alkaline phosphatase, BSP-2 = Bone sialoprotein-2, MTA = mineral trioxide aggregate.

View larger version (30K): [in a new window]   Figure 4. Effects of root-end filling material on alkaline phosphatase activity. Alkaline phosphatase activity was measured colorimetrically for PDL fibroblasts and gingival fibroblasts (GF) grown in the presence of plastic (solid diamonds), MTA (solid squares), HICR (solid triangles), and bone particles (circles) for from 1 to 17 days in culture. Enzyme activity was compared for PDL fibroblasts (A,C) and gingival fibroblasts (B,D) cultured in the presence of fresh (C and D) and washed (A and B) root-end filling materials. Note that the PDL fibroblasts expressed approximately twice the alkaline phosphatase activity of gingival fibroblasts. Each point represents the mean of 4 samples. Error bars represent standard deviation from the mean. ANOVA analysis was applied to all the values, and statistical significance was determined at p < 0.01. Data points represent a typical experiment that was repeated in triplicate.