Effect of sodium tanshinone IIA sulfonate, an ectosteric inhibitor of cathepsin K, on osteogenic differentiation of pulp cells and on the development of apical periodontitis

Abstract

Apical Periodontitis (AP) is characterized by the presence of inflammation and destruction of mineralized and non-mineralized periapical tissues, processes that involve the production of cytokines, osteoclastogenesis and the action of enzymes, including cathepsin K (CatK). Tanshinones, substances extracted from the plant Salvia miltiorrhiza, are widely used in traditional Chinese Medicine to treat cardiovascular, and osteolytic diseases, such as Osteoporosis, among many others. Sodium tanshinone IIA sulfonate (T06), a water-soluble derivative of tanshinone IIA (TIIA), inhibits bone resorption via selective inhibition of collagenolytic activity of CatK (ectosteric CatK inhibition), without interfering with other active sites of the enzyme and does not affect osteoclastogenesis; these are two important/promising properties of bone antiresorptive. Another factor to be considered is that TIIA induces osteogenic differentiation of mesenchymal stem cells, but it is not known whether T06 has this property. The aims of the study are (1) to evaluate the effect of T06 on inflammation and bone loss in experimentally induced AP in mice and (2) to evaluate the cytocompatibility and effect of T06 on the proliferation, migration, osteogenic and odontogenic differentiation of cells of the human dental pulp (hDPCs) in vitro. For the in vivo study, 40 male C57BL/6 mice will be distributed into 4 groups (n=10/group): group with AP induction and treatment with T06; group with AP induction and treatment with T06 vehicle (distilled water); group without AP induction and treatment with T06; group without AP induction and treatment with distilled water. For AP induction, coronal opening and pulp removal from the two mandibular first molars will be performed, and the pulp chamber will remain open to the oral environment for 30 days. The substances will be administered by gavage, once a day, during the 30 days of the experiment. After euthanasia, a hemi-mandible will be submitted to microtomographic examination to evaluate the volume of periapical bone loss and, then, to histotechnical processing for analysis of inflammation and quantification of osteoclasts in the periapical region. In the remaining hemi-mandible, the roots of the first molar and the adjacent bone will be collected and prepared for analysis of the messenger RNA expression of genes related to inflammation and osteoclastogenesis, using quantitative real-time polymerase chain reaction (RT- qPCR). For the in vitro study, hDPCs exposed to T06 and not exposed (control) will be evaluated using different methodological approaches such as viability, apoptosis, proliferation, migration, and osteogenic and odontogenic differentiation analysis. The statistical tests will be chosen according to the distribution and homoscedasticity of the data, using a significance level of 5%. (AU)