Topical application of bFGF on acid-conditioned and non-conditioned dentin: effect on cell proliferation and gene expression in cells relevant for periodontal regeneration

Abstract

Material and methods: Periodontal regeneration is still a challenge in terms of predictability and magnitude of effect. In this study we assess the biological effects of combining chemical root conditioning and biological mediators on three relevant cell types for periodontal regeneration. Bovine dentin slices were conditioned with 25% citric acid followed by topical application of basic fibroblast growth factor (bFGF, 10 and 50 ng). We used ELISA to assess the dynamics of bFGF release from the dentin surface and RT-qPCR to study the expression of Runx2, Col1a1, Bglap and fibronectin by periodontal ligament (PDL) fibroblasts, cementoblasts and bone marrow stromal cells (BMSC) grown onto these dentin slices. We also assessed the effects of topical application of bFGF on cell proliferation by quantification of genomic DNA.

Results: Acid conditioning significantly increased the release of bFGF from dentin slices. Overall, bFGF application significantly (p<0.05) increased cell proliferation, except for BMSC grown on non-conditioned dentin slices. Dentin substrate discretely increased expression of Col1a1 in all cell types. Expression of Runx2, Col1a1 and Fn was either unaffected or inhibited by bFGF application in all cell types. We could not detect expression of the target genes on BMSC grown onto conditioned dentin.

Conclusion: Acid conditioning of dentin improves the release of topically-applied bFGF. Topical application of bFGF had a stimulatory effect on proliferation of PDL fibroblasts, cementoblasts and BMSC, but did not affect expression of Runx2, Col1a1, Bglap and fibronectin by these cells.

Figure 1. Concentration of bFGF in acqueous-based cell culture medium, after 10 to 480 min of incubation. 50 ug/mL of bFGF was applied topically to dentin slices with and without previous acid conditioning and the quantity of bFGF in the medium was determined in aliquots of the culture medium by ELISA at the indicated periods. In positive control experiments (without dentin slices), the same concentration of bFGF was added directly to the culture medium. Bars and vertical lines indicate means and standard deviations of three independent experiments, assayed in duplicate (* indicates p<0.05 vs previous period; # indicates p<0.05 vs initial period of assessment)

Figure 2. Genomic DNA quantitation indicating the proliferation of murine periodontal ligament fibroblasts (mPDL, panel “A”), cementoblasts (OCCM, panel “B”) and bone marrow stromal cells (BMSC, panel “C”), according to the experimental conditions. Cells were plated onto dentin slices on which bFGF (10 and 50 ng) was applied topically both with and without previous acid conditioning. Cells were grown in complete culture medium for 24, 48 and 72 h. In positive control samples, cells were plated on tissue culture plastic (no dentin slices) and stimulated with 50 ng/mL of bFGF and genomic DNA collected at the same periods. The different symbols indicate mean and the vertical lines indicate the standard deviation of genomic DNA quantity from three independent experiments, measured in triplicate at each time point (* indicates p<0.05 control vs 50 ng/mL bFGF, # indicates p<0.05 control vs 10 ng/mL bFGF, and ! indicates p<0.05 10 ng/mL bFGF vs 50 ng/mL bFGF)

Figure 3. Relative changes in gene expression (mRNA) of Runx2, Col1a1, Fn and Bglap by cementoblasts (OCCM, panel “A”), periodontal ligament fibroblasts (mPDL, panel “B”) and bone marrow stromal cells (BMSC, panel “C”) cultured on different substrates: tissue culture-treated plastic, non-conditioned (NC) and conditioned (C) dentin slices. The same quantity of cells (5×104) was seeded onto the different substrates and total RNA collected after 24 hours. Gene expression of the indicated target genes was determined by RT-qPCR. Bars indicate the mean result of the relative change (fold regulation) of normalized target gene expression in comparison with vehicle-treated samples in each substrate. Bars indicate mean and vertical lines indicate standard deviation of a minimum of three independent experiments