Calcitonin Gene-related Peptide Expression in Human Periodontal Ligament

An experimental study was performed according to Colombian Ministry of Health recommendations regarding ethical issues in research involving human tissue. Written informed consent was obtained from each of the patients participating in the study (18-27 years old, healthy, not medicated, and nonsmoking human donors, with premolars extraction indicated for orthodontic reasons). All teeth used were caries- and restoration-free with complete root development determined radiographically (and confirmed visually after extraction), without signs of periodontal disease or traumatic occlusion, and without previous orthodontic forces.

Experimental procedure Periodontal ligament samples were obtained from 90 premolars that were randomly divided into 3 groups of 30 premolars each, as follows: I) Untreated teeth control group (without orthodontic forces); II) Moderate force group; III) Severe force group. Teeth in moderate and severe orthodontic force groups were submitted to tipping and extrusion orthodontic movements.

Orthodontic forces were applied following the exact same methodology that was used in a previous study (15), in which, prior to orthodontic force application, the occlusal surface of the first mandibular molar was raised with a block of resin (Filtek Z350, 3M Espe, Seefeld, Germany) until the premolars were out of occlusion. A convertible standard buccal tube (Orthorganizer, Carlsbad, CA) was bonded over the buccal face of the first molar with resin (Light Bond, Reliance Orthodontic Products Inc, Itasca, IL). An MBT slot size 0.022 bracket (Ref. 702-393 MC, Orthorganizer) was bonded over the buccal face of the premolars. One 0.0017 x 0.025 in titanium molybdenum alloy (TMA) wire cantilever was inserted into each first molar tube and the wire was bent buccally to form a helix.

The cantilever was clinched to the distal end of the tube and exerted a tipping and extrusive force on the premolar. For the teeth in the moderate force group, the activation angle was 45º with a force of 56 g. For the severe force group, the activation angle was 90º with a force of 224 g. For both groups, forces were measured with an orthodontic dynamometer. Once the force was measured, the free-end of the sectional arch was hooked to the bracket with a metallic ligature. Seven days after, the ligature, the sectional arch, the tube, and the resin block were removed in order to perform the extraction procedure.

All teeth were anesthetized with 1.8 mL 4% prilocaine without vasoconstrictor by infiltrative injection for upper premolars and inferior alveolar nerve block injection for lower premolars. Adequate pulpal anesthesia was ascertained with a negative response to an electronic pulp vitality test.

Sample collection Teeth in the control and orthodontic forces group were extracted 10 min after anesthetic application with conventional methods and without excessive injury to the periodontal ligament. Immediately after extraction, PDL samples were obtained from the entire length of the root with a periodontal curette, placed on an Eppendorf tube, snap-frozen in liquid nitrogen, and kept at -70°C until use.

Radioimmunoassay (RIA) PDL samples were defrosted without thermal shock, dried on a filter, and individually weighed on an analytical balance. Neuropeptide was extracted by adding 150 µL of 0.5 mol L-1 acetic acid and double boiling in a thermostat bath for 30 min in accordance with previously reported protocols (9,12,15,18-21).

CGRP expression was determined by competition binding assays using a human CGRP-RIA kit from Phoenix Peptide Pharmaceutical (Ref. RK-015-02, Belmont, CA). Fifty µL of each sample solution were incubated in polypropylene tubes at room temperature for 20 h with 100 µL of primary antibody and 100 µL of different CGRP concentrations (10 pg mL-1 -1280 pg mL-1). Then, 50 µL of 125I-CGRP was added and left incubate for another 24 h. Bound fractions were precipitated by the addition of 100 µL of a secondary antibody (Goat Anti- Rabbit IgG serum), 100 µL of normal rabbit serum, and 500 µL of RIA buffer containing 1% polyethylene glycol 4000. After 2 h of incubation at room temperature, tubes were spun at 3000 rpm for 45 min at 4º C. The supernatants were decanted, and pellet radioactivity was read on a Gamma Counter (Gamma Assay LS 5500; Beckman, Fullerton, CA). Standard curves of authentic peptide were made in buffers identical to the tissue extracts on semi-log graph paper.

Finally, analysis of the binding data assessed the amount of CGRP present in every sample, using the percentage of maximum binding (B/B0%) calculated for each unknown sample, reading across the graph to the point of intersection with the calibration curve, where the corresponding X-axis coordinate is equivalent to the concentration of peptide in the assayed sample.

Statistical analysis Values are presented as CGRP concentration in pmol per mg of PDL. Mean, standard deviation, medians, and maximum/minimum values are presented for each group. Kruskal Wallis test was performed to establish statistically significant differences between groups (P<0.05). LSD posthoc comparisons were also performed.