Fibrin clot adhesion to conditioned root surfaces: an in vitro study with scanning electron microscopy analysis

Aim: Periodontal regeneration is contingent on the adhesion and maturation of fibrin clot to a root surface exposed to periodontal disease. Root surface demineralization in vitro improves the formation of a stable union between the fibrin clot and the root surface. In scientific literature there are not studies of comparison that stand which demineralizing agent is the best in promoting fibrin clot adhesion. The aim of this study was to evaluate and compare the efficacy of six root conditionings in removing the smear layer and developing the fibrin clot in static and dynamic conditions. Methods: 36 single-root teeth extracted for periodontal disease were cut with a microtome in order to obtain 72 specimens that were divided in three groups: 24 samples not covered with blood, 24 covered with fresh human whole blood, 24 covered with blood and rinsed in a rotary shaker table (Vortex®). 4 specimens from each group were conditioned for 3 minutes with: physiological saline solution (FISIO) as the control group, saturated solution of citric acid 25% (AC), ethylenediaminetetraacetic acid 24% (EDTA), a solution of tetracycline 200mg/mL (TETRA), a solution of tetracycline and citric acid (TETRA+AC), Prefgel® and successively Emdogain® (EMD). Then the 48 samples from group 2 and 3 were covered with blood, which was allowed to coagulate for 20 minutes in a 37°C chamber. The blocks were rinsed and dehydrated under standardized conditions; specimens of group 3 were vortexed (100rpm). All the blocks were then sputtered with gold and analyzed with SEM. SEM images were evaluated by two blinded examiners, starting from the cementoenamel junction (CEJ), at 5 standardized points 2 mm distant from each other. A statistical analysis was performed. Results: EMD samples showed a more disorganized smear layer, in which is probably present the residual vehicle (propylene glycol alginate). However, in the 90% of all the specimens smear layer was found. AC treated samples showed a firmly adherent fibrin clot that covered the surfaces for the 70% of the specimens (the data was statistically relevant). Same results were found in TETRA+AC samples. The conditioning with EDTA, TETRA and EMD resulted in a sparsely organized clot worsened by the application of tensile forces, especially in TETRA samples. Only few blood cells without any clot organization were found in the control group, confirming that conditioning root surfaces improves the fibrin clot adhesion. Conclusion: The best formation of fibrin clot was observed for AC treated samples; this is probably due to the increase of root surface wettability caused by AC. The other root conditioning agents, even if lead to results better than the control group, are more susceptible to external forces and do not promote a stable fibrin clot adhesion.