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.

The smear layer created by scaling and root planing is physiologically eliminated in a biphasic process

Mechanical instrumentation of the root surface causes the formation of a smear layer, which is a physical barrier that can affect periodontal regeneration. Although different procedures have been proposed to remove the smear layer, there is no information concerning how long the smear layer persists on root surfaces after instrumentation in vivo. This study assessed the presence of the smear layer on root surfaces over a 28-day period after subgingival instrumentation with hand instruments. Fifty human teeth that were referred for extraction because of advanced periodontal disease were scaled and root planed (SRP) by a single experienced operator. Ten teeth were randomly assigned to be extracted 7, 14, 21, and 28 days after SRP. Another 10 teeth were extracted immediately after instrumentation (Day 0, control group). The subgingival area of the instrumented roots was evaluated with scanning electron microscopy. Representative photomicrographs were assessed by a blinded and calibrated examiner according to a scoring system. A rapid and significant (p < 0.05, Z test) initial reduction in the amount of smear layer was observed at 7 days, and a further significant (p < 0.05) decrease was observed 28 days after SRP. Interestingly, even 28 days after SRP, the smear layer was still present on root surfaces. This study showed that the physiological elimination of the smear layer occurred in a biphasic manner: a rapid initial reduction was observed 7 days after instrumentation, which was followed by a slow process leading to a significant decrease 28 days after instrumentation.

Effect of acid fruit juices combined with electric or sonic toothbrushing on root dentin permeability: an in vitro study

The purpose of this in vitro study was to quantify the alterations on human root dentin permeability after exposure to different acid fruit juices and to evaluate the effect of toothbrushing with electric or sonic toothbrush after acid exposure. The root dentin of 50 extracted third molars was exposed with a high speed bur. Crowns were sectioned above the cementoenamel junction and root fragments were used to prepare dentin specimens. Specimens were randomly assigned to 5 groups according to the fruit juice (kiwifruit, starfruit, green apple, pineapple and acerolla). Each specimen was connected to a hydraulic pressure apparatus to measure root dentin permeability using fluid filtration method after the following sequential steps: I) conditioning with 37% phosphoric acid for 30 s, II) root scaling, III) exposure to acid fruit juices for 5 min and IV) electric or sonic toothbrushing without dentifrice for 3 min. Data were analyzed statistically by the Wilcoxon and Mann-Whitney tests at 5% significance level. All fruit juices promoted a significant increase of dentin permeability while toothbrushing decreased it significantly (p<0.05). It may be concluded that all acid fruit juices increased root dentin permeability, while toothbrushing without dentifrice after acid exposure decreased the permeability. The toothbrush mechanism (electric or sonic) had no influence on the decrease of root dentin permeability.

O objetivo deste trabalho in vitro foi quantificar as alterações na permebilidade da dentina radicular humana após exposição a diferentes sucos de frutas ácidas e avaliar o efeito da escovação, com escova elétrica ou sônica, após a exposição ácida. A dentina radicular de 50 terceiros molares foi exposta com a utilização de fresas em alta rotação. As coroas foram seccionadas acima da junção cemento-esmalte e apenas os fragmentos radiculares foram utilizados no preparo dos espécimes. Os espécimes foram aleatoriamente divididos em 5 grupos de acordo com o suco de fruta aplicado (kiwi, carambola, maça verde, abacaxi e acerola). Cada espécime foi conectado a um aparelho de pressão para medir a permeabilidade dentinária por meio do método de filtração de líquidos após as seguintes etapas sequências: I) condicionamento com ácido fosfórico 37% durante 30 s, II) raspagem da raiz, III) exposição aos sucos de frutas por 5 min, IV) escovação com escova elétrica ou sônica durante 3 min. Os dados foram analisados estatisticamente pelos testes Wilcoxon e Mann-Whitney com nível de significância de 5%. Os resultados mostraram que todos os sucos de frutas testados promoveram aumento significativo da permeabilidade dentinária e os procedimentos de escovação causaram diminuição. Pode-se concluir que os sucos de frutas ácidas aumentaram a permeabilidade da dentina radicular, enquanto que a escovação sem dentifrício imediatamente após a exposição ácida promoveu redução da permeabilidade. Além disso, o mecanismo da escova (elétrica ou sônica) não teve influência na redução da permeabilidade dentinária.

Condicionamento com ácido cítrico e os efeitos biológicos na aplicação tópica de bFGF e BMP-7 em células relevantes para a regeneração periodontal / Conditioning with citric and biological effects of topical bFGF and BMP-7 in relevant cells to periodontal regeneration

A regeneração periodontal ainda representa um importante desafio, devido a limitações na efetividade e imprevisibilidade das abordagens clínicas. O condicionamento radicular e a utilização de mediadores biológicos, como os fatores de crescimento, são duas das técnicas que tem sido estudadas para esta finalidade; também tem sido proposta a combinação de ambas. Foi avaliado in vitro o efeito do condicionamento ácido da dentina na liberação de bFGF e BMP-7 aplicados topicamente de forma isolada ou associados, além da influência de tais tratamentos na expressão de genes-alvo relacionados ao metabolismo de tecido conjuntivo mineralizado e não mineralizado em três tipos celulares relevantes para o processo regenerativo no microambiente periodontal. Espécimes de dentina bovina foram subdivididos em dois grupos: não condicionados e condicionados com ácido cítrico 25% durante 3 minutos. bFGF recombinante humano (10 e 50 ng), BMP-7 (100 e 300 ng) e associação bFGF/BMP-7 (50 ng e 100 ng, respectivamente) foram suspendidos em 50 µL de meio de cultura e aplicados topicamente sobre a dentina. A liberação de fatores de crescimento aplicados topicamente sobre a dentina condicionada e não condicionada foi determinada por meio de ELISA. Linhagens celulares de camundongos – fibroblastos do ligamento periodontal, cementoblastos e células do estroma ósseo – foram plaqueadas sobre os espécimes de dentina com e sem condicionamento ácido prévio, tratados ou não com os fatores de crescimento. Após 24 horas, as células aderidas sobre as amostras de dentina foram coletadas, o RNA total extraído e a expressão gênica de colágeno 1-alfa1, fibronectina e Runx2 foi avaliada por RTqPCR. Os resultados encontrados neste estudo indicam que os fatores de crescimento são retidos pela dentina após aplicação tópica com o pico de liberação em 2 horas; o condicionamento ácido da dentina favoreceu a retenção dos fatores de crescimento aplicados topicamente sobre a dentina; e os três tipos celulares avaliados respondem diferentemente a uma mesma condição experimental; e que a aplicação tópica dos fatores de crescimento sobre a dentina atenuou os efeitos dos mesmos fatores utilizados diretamente no meio de cultura, sendo esta atenuação menos marcante em amostras previamente condicionadas com ácido cítrico

Periodontal regeneration still poses a challenge both in terms of predictability and magnitude of effect. Chemical root conditioning and the use of biological mediators are two strategies studied to improve the results of regenerative therapies. The objective of this study was to determine the effect of acid conditioning of dentin on the release of topically applied bFGF and BMP-7, isolated or associated. We also evaluated the impact of such treatment in the expression of target genes related to the metabolism of mineralized connective tissue and non-mineralized in three cell types relevant for the regenerative process in the periodontal microenvironment. Dentin specimens, which were obtained from bovine teeth, were divided into two groups: non-conditioned and conditioned with 25% citric acid for 3 minutes. Human growth factors produced by recombinant DNA technology were diluted in 50 mL of culture medium and topically applied on the dentin in the following amounts: recombinant human bFGF (10 and 50 ng), BMP-7 (100 and 300 ng) and associated bFGF/BMP-7 (50 ng and 100 ng, respectively). The released of growth factor topically applied on the conditioned and non-conditioning dentin samples was determined by ELISA. Murine cell lines - periodontal ligament fibroblasts, cementoblasts and bone marrow stromal cells - were grown on dentin specimens with and without prior acid conditioning and application of growth factors. After 24 hours, the cells adhered on the samples of dentin were collected, total RNA extracted and gene expression of collagen 1-alpha 1, fibronectin and Runx2 was determined by RT-qPCR. The results of this study indicate that: growth factors are retained by dentin after topical application and that the peak release occurs in two hours; the conditioning of dentin with citric acid favored the retention of topically applied growth factors to dentin; all three cell types respond differently to the same experimental condition evaluated; the topical application of growth factors on dentin attenuated the effects of these factors used directly in the culture medium, and this attenuation was less marked in the samples previously conditioned with citric acid