This experimental study was conducted following the recommendations of the Colombian Ministry of Health regarding ethical issues of scientific research involving human tissues. It was approved by the Bioethics Committee of the Faculty of Dentistry at the Universidad Cooperativa de Colombia in Pasto (CBE07). Written informed consent was obtained from all patients participating in this study. This study was registered in clinicaltrials.gov under the title: “Importance of Neurogenic Inflammation in the Angiogenic Response of the Dental Pulp as a Defensive Response”, trial registration number NCT03804034, dated 01/15/2019, URL: https://clinicaltrials.gov/ct2/show/NCT03804034?term=NCT03804034&draw=2&rank=1
Human dental pulp was obtained from 20 freshly extracted maxillary and 20 mandibular premolars with orthodontic indications from 20 healthy patients, 8 men and 12 women, aged between 18 and 30 years. Patients taking medications for pain, smokers, and pregnant women were excluded. All the teeth included in this study had a normal masticatory function with a clinically and radiographically complete root development, without caries, restorations, orthodontic forces, periodontal disorders, or parafunctional habits.
The sample size was calculated with the TAMAMU 1.1® program (Tokio, Japan) based on the mean and standard deviation of the variables measured in previous studies [18, 19], using a 95% confidence interval and a sample power of 80%, to obtain a total sample size of 20 patients (5 per group). Patients were randomly assigned (Random.org App, Randomness and Integrity Services Ltd, Dublin, Ireland) to each group for a total of 10 premolars (5 upper and 5 lower from the same side of each patient) per group: a) Control group (5 upper and 5 lower healthy premolars from the same side); b) Occlusal trauma (5 upper and the 5 lower premolars that were in contact under occlusal trauma); c) Orthodontic force (5 upper and 5 lower premolars); d) Occlusal trauma and orthodontic force (5 upper and 5 lower premolars that were in contact under occlusal trauma and orthodontic force). Contralateral teeth were also extracted for orthodontic reasons but were not used in the study. (Fig. 1).
In the occlusal trauma group (Fig. 1b), an occlusal interference was placed on the mandibular premolars. An articulating paper was used to mark the contact area between the upper and mandibular premolars indicated for extraction, following the study model reported by Caviedes Bucheli et al. [18]. Lower premolar was acid-etched with 37% phosphoric acid (SuperEtch; SDI Ltd, Bayswater, Victoria, Australia) for 15 seconds. After washing and drying, a bonding agent (STAE; SDI Ltd) was applied and light-cured for 15 seconds, and finally a 2mm resin block (Filtek Z350; 3M Espe, Seefeld, Germany) was placed and light-cured (40 sec) over the contact area of the upper premolar. An articulating paper was used to verify that only the premolars indicated for extraction had contact during normal occlusion as well as in lateral movements. Patients were given chewing gums and instructed to repeat 20 masticatory cycles for 30 sec, followed by a 30 sec rest interval, and repeat the sequence for 30 min. This chewing cycle was repeated three times every 8 h for the first 24 h and it was confirmed by each patient in a format with the established schedules.
For the moderate orthodontic force group (Fig. 1c), the occlusal surface of the first mandibular molar was raised using a block of resin (Filtek Z350; 3M Espe, Seefeld, Germany) until the premolars were out of occlusion before applying an orthodontic force, following the study model reported by Caviedes-Bucheli et al. [19]. Convertible standard buccal tubes (Orthorganizer, Carlsbad, CA) were bonded on the buccal face of the first upper and lower molar with resin (Light Bond; Reliance Orthodontic Products Inc, Itasca, IL). A McLaughlin, Bennett, and Trevisi (MBT) slot size 0.022 bracket (Ref. 702-393 MC, Orthoorganizer) was bonded over the buccal face of the upper and lower first premolars. A 0.0017/0.025 titanium molybdenum alloy (TMA) wire cantilever was inserted into each first molar tube and the wire was bent buccally to form a helix with an activation angle of 45°. The cantilever was clinched to the distal end of the tube, and the orthodontic force was measured with an orthodontic dynamometer to 56g exerting a tipping and extrusive force on the premolar for 24 h before being extracted.
In the occlusal trauma and moderate orthodontic force group (Fig. 1d), occlusal interferences combined with orthodontic force were performed as previously described; this force was applied to the teeth for 24 h before being extracted, without placing the resin bumpers in the posterior molars.
Sample Collection
All the teeth included in this study were anesthetized with 1.8 ml of 4% prilocaine without a vasoconstrictor (Pricanest, Ropsohn Therapeutics Ltd.) by infiltrative injection for maxillary premolars and inferior alveolar nerve block injection for mandibular premolars. The teeth were extracted 5 min after administering the anesthetics with conventional methods. Immediately after the extraction, the teeth were sectioned using a Zekrya bur (Dentsply, Tulsa, OK) in a high-speed handpiece irrigated with saline solution. The pulp tissue was obtained using a sterile endodontic excavator, placed on an Eppendorf tube, snap frozen in liquid nitrogen, and kept at −70 °C until the radioimmunoassay test was performed.
Radioimmunoassay (RIA)
Tissue samples were defrosted without thermal shock, dried on a filter, and weighed on an analytic balance. Peptides were extracted by adding 150 μL of 0.5 M acetic acid and double boiled in a thermostat bath for 30 min.
SP expressions (Phoenix Peptide Pharmaceutical RK-061-05) and CGRP (Phoenix Peptide Pharmaceutical RK-015-02) were determined by competitive binding assays using the corresponding kits for each substance following previously published protocols [18-20]. The VEGF expression was equally determined by competition binding assays using a human, recombinant VEGF165 (G143AB; Genentech, Inc., Los Angeles, CA, USA), primary antibody, polyclonal rabbit antiserum to VEGF165 (27906-17, Genentech, Inc., Los Angeles, CA, USA), and human recombinant 125I-VEGF tracer (NEX328, NEN Life Science Products, Inc. Boston, MA, USA). Each sample solution (50 μL) was incubated in tubes at 18 °C for 20 h with 100 μL of primary antibody, and 100 μL of different peptide concentrations (SP: 7.42 × 10-3-0.89 pmol/mL; CGRP: 2.64 × 10-3-0.32 pmol/mL; VEGF: 2.5 × 10-4-3.2 x 10-2 pmol/mL) was added with 50 μL of radiolabeled factors and incubated for 24 h. Bound fractions were precipitated with secondary antibody (100 μL), 100 μL of fetal bovine serum, and 500 μL of RIA buffer containing 1% prolyethylene glycol 8000. After 2-h incubation at 20 °C, the tubes were centrifuged at 3,000 rpm for 45 min at 4 °C. The supernatants were decanted, and the pellet radioactivity was read in a gamma counter. The peptide concentration was determined according to calibration curves of the radioactivity in each mixture. A blinded operator analyzed the treatment effects applied to the teeth in the pulp tissue samples. The samples were coded so blinded operator could not know what stimuli was applied on the tissue being analyzed.
Statistical Analysis
Results are presented as SP, CGRP, and VEGF expressions in pmol/mg of pulp tissue. Means and standard deviations, as well as minimum and maximum values for each neuropeptide and VEGF, were calculated. A Kolmogorov-Smirnov (KS) test was used to determine if the results obtained for each peptide adjust to normality, in order to perform an analysis of variance (ANOVA) test to determine statistically significant differences, followed by Tukey’s HSD post-hoc and Bonferroni correction tests at a significance level of p< 0.05.