The Effect of Three Different Rotary Instrumentation Systems on Substance P and Calcitonin Gene-related Peptide Expression in Human Periodontal Ligament Javier Caviedes-Bucheli, DDS, MSc,* Maria Mercedes Azuero-Holguin, DDS,* Luisa Gutierrez-Sanchez, DDS,* Ferelba Higuerey-Bermudez, DDS,* Veronica Pereira-Nava, DDS,* Nelson Lombana, PhD,* and Hugo Roberto Munoz, DDS, MA † Abstract Introduction: The purpose of this study was to quantify the effect of three different rotary root canal preparation systems on substance P and calcitonin gene-related peptide expression in healthy human periodontal liga- ment. Methods: Fifty periodontal ligament samples were obtained from healthy premolars in which extrac- tion was indicated for orthodontic reasons. Before extraction, 40 of these premolars were equally divided into four groups, and root canals were prepared using four different systems: the ProTaper Universal rotary system, the RaCe rotary system, the Mtwo rotary system, and the hand instrumentation technique. The re- maining 10 healthy premolars that were extracted without treatment served as a negative control group. All periodontal ligament samples were processed, and SP and CGRP were measured by radioimmunoassay. Results: Greater SP and CGRP expression were found in the ProTaper Universal group followed by the hand instrumentation group, the RaCe, and the Mtwo groups. The lower SP and CGRP values were for the negative control group. The Kruskal-Wallis test showed statistically significant differences between groups (p < 0.0001). Post hoc Least Significant Difference (LSD) tests showed statistically significant differences in SP and CGRP expres- sion between the negative control group and all the other groups except the Mtwo group. Hand instrumentation also showed statistically significant differences with all the other groups, except the ProTaper Universal group. Differences between the three rotary systems were also statistically significant. Conclusion: SP and CGRP expression in periodontal ligament increases when teeth are prepared with ProTaper Universal and RaCe rotary instrumentation systems as well as with hand instrumen- tation. Mtwo maintains SP and CGRP levels. (J Endod 2010;36:1938–1942) Key Words Calcitonin gene-related peptide, human periodontal ligament, neurogenic inflamma- tion, rotary instrumentation, substance P A frequent problem in endodontics is the development of post-treatment symptom- atic apical periodontitis, which may vary from a low-intensity sensitivity when biting over the tooth to a severe pain to even the slightest touch (1). Apical periodontitis is defined as a circumscribed inflammation of the periodontal ligament in the apical region and it has been reported to be provoked by extrusion of different irritants from the root canal system (such as dentin debris, necrotic tissue, microorganisms, irrigants and/or filling materials) towards the periapex during canal preparation, generating an antigen-antibody reaction with the correspondent inflammatory reaction, even when working length is well established (2, 3). Similar to dental pulp, periodontal ligament inflammation has a neurogenic source, which is induced by the release of neuropeptides from periapical tissue C- type nerve fibers, after being injured during root canal therapy (4). Substance P (SP) and calcitonin gene-related peptide (CGRP) are capable of triggering vasodilation, plasma extravasation, immune system activation, chemotaxis, recruitment, and/or regu- lation of inflammatory cells such as macrophages, mast cells, and lymphocytes (5). Finally, the release of inflammatory mediators in the periodontal ligament generates vascular stasis in the affected area (6, 7). Recent evidence has suggested that human fibroblasts are able to produce SP and that neuropeptides could also regulate the expression of angiogenic growth factors in fibroblasts, suggesting that these cells also play a role in neurogenic inflammation (8, 9). These biological effects could explain the clinical events of pain and inflammation observed during symptomatic apical periodontitis after root canal therapy (10). It has been reported that the severity of periodontal ligament inflammation is directly proportional to the degree of the tissue damage (ie, the quantity of apically extruded debris [1, 11] and the mechanical stress exerted on the tooth [12]). It also has been shown that all root canal preparation techniques cause some degree of debris extrusion (11, 13–16). However, the amount of apically extruded irritants may vary according to the technique and the characteristics of the instrument used (3). According to manufacturers, nickel-titanium rotary instruments, such as ProTaper Universal (Dentsply Maillefer, Ballaigues, Switzerland), Mtwo (VDW, Munich, Germany) and RaCe (FKG, La Chaux-de-Fonds, Switzerland), have been designed with different physical characteristics (ie, profile section, core diameter, rake angle, From the *Postgraduate Endodontics Department, School of Dentistry, Pontificia Universidad Javeriana, Bogota ´ , Colombia; and † Postgraduate Endodontics Depart- ment, School of Dentistry, Universidad de San Carlos de Guatemala, Guatemala City, Guatemala. Address requests for reprints to Dr Javier Caviedes-Bucheli, School of Dentistry, Pontificia Universidad Javeriana, Cra 7 No 40-62 Building 26, Bogota ´ , Colombia. E-mail address: javiercaviedes@gmail.com 0099-2399/$ - see front matter Copyright ª 2010 American Association of Endodontists. doi:10.1016/j.joen.2010.08.043 Clinical Research 1938 Caviedes-Bucheli et al. JOE — Volume 36, Number 12, December 2010