Calcitonin Gene-related Peptide Expression in Human Periodontal Ligament

May 10, 2021 updated by: Javier Caviedes-Bucheli, Institucion Universitaria Colegios de Colombia

The Effect of Orthodontic Forces on Calcitonin Gene-related Peptide Expression in Human Periodontal Ligament

The purpose of this study was to quantify the effect of moderate and severe orthodontic forces on Calcitonin gene-related peptide expression in the healthy human periodontal ligament.

Methodology: 90 human periodontal ligament samples were obtained from healthy premolars where extraction was indicated for orthodontic reasons. Prior to extraction, teeth were divided into 3 groups of 30 samples each: I) Untreated teeth control group; II) Moderate force group: A 56 g force was applied to the premolars for 7 days; and III) Severe force group: A 224 g force was applied to the premolars for 24 hours. All periodontal ligament samples were processed and CGRP was measured by radioimmunoassay.

Study Overview

Status

Completed

Conditions

Intervention / Treatment

Detailed Description

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.

Study Type

Interventional

Enrollment (Actual)

90

Phase

  • Not Applicable

Contacts and Locations

This section provides the contact details for those conducting the study, and information on where this study is being conducted.

Study Locations

  • Colombia
    • Metropolitana
      • Bucaramanga, Metropolitana, Colombia
        • Universidad Santo Tomás

Participation Criteria

Researchers look for people who fit a certain description, called eligibility criteria. Some examples of these criteria are a person's general health condition or prior treatments.

Eligibility Criteria

Ages Eligible for Study

18 years to 27 years (Adult)

Accepts Healthy Volunteers

Yes

Genders Eligible for Study

All

Description

Inclusion Criteria:

- Premolars extraction indicated for orthodontic reasons with complete root development determined radiographically (and confirmed visually after extraction)

Exclusion Criteria:

  • Medicated patients,
  • Smokers patients,
  • Premolars with caries or restorations,
  • Premolars with signs of periodontal disease or traumatic occlusion,
  • Premolars with previous orthodontic forces

Study Plan

This section provides details of the study plan, including how the study is designed and what the study is measuring.

How is the study designed?

Design Details

  • Primary Purpose: Screening
  • Allocation: Non-Randomized
  • Interventional Model: Parallel Assignment
  • Masking: None (Open Label)

Arms and Interventions

Participant Group / Arm
Intervention / Treatment
No Intervention: Untreated teeth control group
Premolars without orthodontic forces
Experimental: Moderate force group
Premolars were submitted to tipping and extrusion orthodontic movements, the activation angle was 45º with a force of 56 g. Forces were measured with an orthodontic dynamometer. For 7 days
Device: Orthodontic forces
56g force and 224g force
Experimental: Severe force group
Premolars were submitted to tipping and extrusion orthodontic movements, the activation angle was 90º with a force of 224 g. Forces were measured with an orthodontic dynamometer. For 24 hours
Device: Orthodontic forces
56g force and 224g force

What is the study measuring?

Primary Outcome Measures

Outcome Measure
Measure Description
Time Frame
Calcitonin gene-related peptide in periodontal ligament
Time Frame: 24 hours stimulation and 7 days stimulation
CGRP expression change in periodontal ligament
24 hours stimulation and 7 days stimulation

Collaborators and Investigators

This is where you will find people and organizations involved with this study.

Sponsor

Institucion Universitaria Colegios de Colombia

Collaborators

Universidad Santo Tomas

Publications and helpful links

The person responsible for entering information about the study voluntarily provides these publications. These may be about anything related to the study.

General Publications

Study record dates

These dates track the progress of study record and summary results submissions to ClinicalTrials.gov. Study records and reported results are reviewed by the National Library of Medicine (NLM) to make sure they meet specific quality control standards before being posted on the public website.

Study Major Dates

Study Start (Actual)

June 4, 2019

Primary Completion (Actual)

November 7, 2019

Study Completion (Actual)

March 10, 2020

Study Registration Dates

First Submitted

May 5, 2021

First Submitted That Met QC Criteria

May 5, 2021

First Posted (Actual)

May 11, 2021

Study Record Updates

Last Update Posted (Actual)

May 13, 2021

Last Update Submitted That Met QC Criteria

May 10, 2021

Last Verified

May 1, 2021

More Information

Terms related to this study

Keywords

Other Study ID Numbers

  • JCB003

Plan for Individual participant data (IPD)

Plan to Share Individual Participant Data (IPD)?

NO

Drug and device information, study documents

Studies a U.S. FDA-regulated drug product

No

Studies a U.S. FDA-regulated device product

No

This information was retrieved directly from the website clinicaltrials.gov without any changes. If you have any requests to change, remove or update your study details, please contact register@clinicaltrials.gov. As soon as a change is implemented on clinicaltrials.gov, this will be updated automatically on our website as well.

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