Gingival crevicular fluid and root resorption

In our knowledge, very few studies exist on the association between GCF components’ level and root resorption during orthodontic movement. Recently, the dentine phosphoproteins (DPP, non-collagenous proteins of dentin matrix) and the dentine sialoprotein (DSP) were detected in GCF: the levels of these molecules were found to be higher in primary teeth undergoing physiologic root resorption and in teeth with orthodontically induced severe root resorption as compared to non-resorbing controls (203-205). The role of cytokines of the RANKL/RANK/OPG system in inducing bone remodeling during orthodontic tooth movement has been demonstrated: higher expression of RANKL was found in the compression side, whereas higher expression of OPG was found in the tension side. Interestingly, the compressed periodontal ligament cells obtained from tissues with severe external apical root resorption may produce large amounts of RANKL (206). In GCF the expression of these molecules has been mainly studied in relation to periodontal disease (189, 190). What remains to be elucidated is whether the RANKL /OPG system may provide an important link between bone remodeling, orthodontic tooth movement and root resorption during orthodontic tooth movement.

During the experimental tooth movement described in chapetrs 6 and 7, the GCF was collected at the buccal/labial and palatin/lingual sites of the first or second premolars in the 4 quadrants that were planned to be extracted at the end of the

experimental period. The collection was made by means of durapore filter membranes.

Briefly, after isolation of the sites from saliva, a first durapore strip was inserted 1mm into the sulcus and left in place for 15sec. After removal of the first strip and after waiting 3 min, a second durapore strip was inserted in the same site, in the same manner. The 2 strips were placed into a microcentrifuge tube and immediately frozen at –70⁰C until the day of the analysis. In case of visible contamination with blood, the strips were discarded. A total of 104 samples were collected in each patient, during the entire study.

We have started the analysis of four markers in the GCF samples of 30 patients who participated and completed the study:

- Receptor activator of NFkB ligand (RANKL) - Osteoprotegerin (OPG)

- Osteopontin (OPN) - Osteocalcin (OC)

On the day of the analysis, 60 µl of phosphate-buffered saline (PBS, pH 7.2) is added to the tubes containing the strips. The strips are gently shaken for 1 minute and then centrifuged at 2000xg for 5 minutes, with the strips kept at the collar of the tube in order to elute GCF components.

Gingival crevicular fluid samples are analyzed by the Bio-Plex 200 Suspension Array System provided by Bio-Rad. The system is a flow-based dual-laser system for simultaneously identifying and quantifying up to 100 different analytes in a single biomolecular assay (xMAP technology). The system detects and measures

molecules bound to the surfaces of fluorescent microspheres, thus providing highly accurate analysis of serum, culture media and other biological samples as small as 25 µl. To perform a multiplex reading, samples are mixed with conjugated

microsphere and reactant mixtures, and fluorescent reporter molecules are added.

The assays are loaded into the well of a 96-well microtiter plate and the plate is inserted into the microplate platform. The platform and array read are controlled by a computer running Bio-Plex Manager software. The detection system in the array reader uses two lasers to analyze the microspheres in the system. The first laser identifies each microsphere and its associated analyte, and the second measures the amount of analyte in the sample.

The principal advantage of the system is that a large number of markers in a small sample can be quantified simultaneously. The same volume would only be sufficient for the analysis of a single marker by ELISA. This can be translated into substantial savings in the cost of reagents and time to perform the assay completely. After strip removal, the supernatant is divided into two aliquots for the determination of the biochemical compounds. One aliquot (25µl) serves for the determination of RANKL by using a single plex assay kit and the other aliquot serves for the simultaneous determination of OPG, OPN and OC by using a three-plex assay kit. Four captured

antibodies for the specific markers are coupled to specific bead sets with different fluorophores. Using multiple bead sets, 4 different assays are simultaneously performed and detected on two aliquots of each individual sample.

At present, it is not possible for the clinicians to predict root resorption with confidence, to prevent or reduce its occurence and also to reverse the destruction of tooth structure during orthodontic treatment. This is especially important for high risk patients where severe root resorption occurring during orthodontic treatment, may result in the total loss of tooth in treatment. The results of the ongoing study may help: i) to better understand the physiopathologic mechanism that takes place during orthodontic movement, responsible of the root resorption ii) to elucidate if one or combination of several markers in crevicular fluid could help us to identify a patient at risk for extensive root resorption during orthodontic treatment iii) to detect root

resorption before the radiographic observation and eventually to avoid in the future the radiography as method for diagnostic screening of the presence of root

resorption.