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 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. Material and Methods: 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.


Introduction
Evidence indicates that current cause-related periodontal therapy is effective in stopping disease progression and reducing local inflammation, but regeneration of periodontal tissues lost as a consequence of disease still poses a challenge both in terms of predictability and magnitude of effect 5. Several studies 2,17,24 have evaluated the potential of a host of biological and chemical mediators, particularly growth and differentiation factors, to improve the results of regenerative therapy. Acid root conditioning is one of intent 4,7 . There is great controversy in regard to the conditioning agents and methods of application.
In fact, there are products currently available commercially for applications in periodontal regeneration based on the biological properties of growth factors. One such product includes in its surgical procedure protocol acid conditioning of the root surface to remove smear layer from the dentin 4 and there is evidence indicating that chemical conditioning with EDTA improved PDGF-induced adhesion of periodontal PDGF without previous EDTA conditioning 3 .
Chemical conditioning of dentin has also been extensively studied and may favor the repair/ on dentin surface induced by chemical conditioning, including removal of smear layer, greater exposure of 8,16 may favor the adsorption of topically applied growth factors and also turn the dentin surface more conductive to cell attachment to promote blood clot attachment.
Our research group has systematically evaluated the most commonly used conditioning agents (citric acid, tetracycline hydrochloride and EDTA), optimizing conditions for their topical use based on the effectiveness of smear layer removal, exposure of cells 6,11,16,23 . These in vitro studies indicated that citric acid is the best candidate for a chemical conditioning agent to improve removal of smear layer, exposure conditioning include: 1) the exposure of endogenous growth factors and biological mediators stored in the dentin or cementum matrix; and 2) the increased adsorption of exogenous growth factors topically applied to the root surfaces due to the exposed There are reports of promising results for periodontal regeneration with the use of root conditioning in pre-clinical in vivo models, but the results of clinical studies using root conditioning as a sole technique or even combined with other techniques are, in general, disappointing 1,18,28 . However, very few studies have assessed the combination of chemical root/dentin conditioning with topical application of biologically-active mediators. Polypeptide growth factors may act locally or systemically and modulate several cell functions, including proliferation, migration, chemotaxis, differentiation, and gene expression 12 . factor (bFGF) is a growth factor with biological effects that may have important implications for periodontal regeneration, namely induction of angiogenesis, cell proliferation and differentiation, resulting in increased extracellular matrix production 15 . bFGF may be useful for periodontal regeneration, because of its angiogenic properties and also the chemotactic and proliferative effects on PDL cells, which may improve healing and regeneration processes. Both pre-clinical studies 19,21,25 and one clinical study 14 assessed the use of bFGF in various concentrations and modes of application, suggesting that bFGF may enhance periodontal regeneration. This study addresses the hypothesis that root conditioning improves the biological effect of bFGF topically applied onto the dentin surface.

Material and methods
Preparation of dentin slices Rectangular dentin slices of 1.0x0.5x0.3 cm (length x width x heigth) were obtained manually from the roots of bovine incisors, by sectioning 2-3 mm apically to the cementum enamel junction with a diamond disc mounted on a slow speed dental handpiece. The portion of the dentin facing the pulp used at low speed. The external surface (i.e., facing the periodontal ligament and bone) of the dentin slices was submitted to 5 scaling strokes by a single operator using a 5-6 Gracey curette. Cells were always seeded onto this external surface (i.e., submitted to Topical application of bFGF on acid-conditioned and non-conditioned dentin: effect on cell proliferation and gene expression in cells relevant for periodontal regeneration the manual scaling) of the dentin slices with an area of 50 mm 2 . After preparation, the dentin slices were then sterilized by ethylene oxide gas to avoid changes to the microstructure of the dentin or accumulation of chemical residues on the dentin surface that could occur by other means of sterilization. containing the desired concentration of the growth factor(s). The 50 μL volume yielded optimal conditions to completely cover the dentin slices. This suspension of DMEM containing the growth factors was removed by aspiration after 2 minutes. Immediately, 50 μL of a 1x10 6 cells/mL suspension of each cell line was pipetted onto the dentin slices and placed in a CO 2 incubator at 37°C for 4 hours for initial cell adhesion. After the initial 4-hour period, 1.5 mL of DMEM supplemented with 2% FBS were added to each well, and they were incubated for a further 20-hour period. In the negative control wells (without dentin slices), the same quantity of cells was plated and the growth factors added directly to the culture medium. Cells were collected mechanically with sterile plastic cell scrapers. The exact same procedure was used to plate the cells for the tissue culture-treated plastic substrate controls in which no dentin slice was used as substrate.

Release of bFGF from the dentin substrate analysis
We prepared dentin slices using the exact same    When bFGF was applied on the dentin substrate, grown on acid-conditioned dentin, but not in nonconditioned dentin; whereas in BMSC cells the dose-dependent, but only in non-conditioned dentin.
However, cementoblasts were less responsive to bFGF in terms of increase in Col1a1 expression, particularly when grown on acid-conditioned dentin. In fact, we cannot rule out a cytotoxic effect in acidconditioned dentin, as the proliferation was assessed DNA integrity or of cell viability by other methods.
It is important to note that in the positive control samples (cells grown directly on tissue culture plastic, without dentin slices) the concentration of bFGF was much greater than in the experimental samples. This of bFGF for the topical application, whereas in the positive control samples (cells grown on tissue culture the same growth factor. We note that it is not possible to "topically apply" or "condition" tissue culture plastic with the growth factor, similarly to what was done with the dentin slices. Also, even though we seeded the cells using the same protocol in both cell culture plastic and dentin slices to assure the exact same n=initial numbers of cells and also a similar cell density, it is possible that the cells grown in tissue culture plastic proliferated faster than those plated onto dentin slices, resulting in different cell numbers at the end of the experimental period. We speculate that this possible the differences in substrate composition rather than by Topical application of bFGF on acid-conditioned and non-conditioned dentin: effect on cell proliferation and gene expression in cells relevant for periodontal regeneration the initial cell numbers and cell density. Anyway, these considerations preclude direct comparison between the results from positive control and experimental groups (cells grown on dentin slices), but the information provided by the negative control samples (treated with the vehicle used to resuspend the bFGF) indicates that bFGF used was biologically active and the information provided by the positive control samples (cells grown on tissue culture plastic and treated with bFGF) informs the general trend for the effects on the expression of the target genes in the different cell types. Nevertheless, we have not determined how much of the initial quantity of the topically applied bFGF was retained/adsorbed on the dentin substrate, eventually present on the dentin surface retained its full biological activity, but we consider the modulation of gene expression and cell proliferation as indirect evidence of bFGF bioactivity.
In summary, we show that previous acid conditioning of dentin improves the release of topically-applied bFGF in aqueous medium. We have also shown that different cell types that are relevant for periodontal regeneration respond differently to bFGF; however there was an overall increase in cell proliferation and in the expression of bone and connective tissue matrix gene expression by cells grown on dentin with bFGF.
Cementoblasts were the most responsive cell type in terms of regulation of gene expression by bFGF in the absence of a dentin substrate; whereas PDL cells were most responsive to bFGF in terms of gene expression when grown on acid-conditioned dentin. Although no clear-cut conclusion may be drawn from our results, the data indicate that topical application of bFGF has biological effects on relevant cell types for periodontal regeneration and warrants in vivo studies to fully assess the potential of this approach on periodontal regeneration.