Clinical Research
Effect of Experimentally Induced Occlusal Trauma on
Substance P Expression in Human Dental Pulp and
Periodontal Ligament
Javier Caviedes-Bucheli, DDS, MSc,* Maria Mercedes Azuero-Holguin, DDS,*
Jose Antonio Correa-Ortiz, DDS,† Marco Vinicio Aguilar-Mora, DDS,*
James Dario Pedroza-Flores, DDS,* Esteban Ulate, DDS,* Nelson Lombana, PhD,*
and Hugo Roberto Munoz, DDS, MA‡
Abstract
Introduction: The purpose of this study was to quantify
the effect of occlusal trauma experimentally induced
with occlusal interferences on substance P (SP) expres-
sion in healthy human dental pulp and periodontal liga-
ment. Methods: Twenty-eight human dental pulp and
periodontal ligament samples were obtained from
healthy premolars in which extraction was indicated
for orthodontic reasons. Before extraction, occlusal
trauma was induced with experimental occlusal interfer-
ences in half of these premolars by placing a resin block
over their occlusal surface and submitting patients to
chew gum for 30 minutes. The remaining healthy
premolars were extracted without occlusal trauma and
served as a control group. All dental pulp and peri-
odontal ligament samples were processed, and SP was
measured by radioimmunoassay. Results: There was
45% and 120% greater SP expression in dental pulp
and periodontal ligament, respectively, of teeth with
experimentally induced occlusal trauma. Paired t test
showed statistically significant differences for both
human dental pulp and periodontal ligament (P = .02
and P < .001, respectively) of teeth submitted to
occlusal trauma when compared with control group
values. Conclusions: SP expression in human dental
pulp and periodontal ligament increases when teeth
are submitted to occlusal trauma experimentally
induced with occlusal interferences. (J Endod
2011;37:627–630)
Key Words
Human dental pulp, neurogenic inflammation, occlusal
trauma, periodontal ligament, substance P
From the *Postgraduate Endodontics Department, School of Dentistry, Pontificia Universidad Javeriana, Bogota, Colombia; †Oral Rehabilitation Department, School
of Dentistry, Pontificia Universidad Javeriana, Bogota, Colombia; and ‡Postgraduate Endodontics Department, School of Dentistry, Universidad de San Carlos de
Guatemala, 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 2011 American Association of Endodontists.
doi:10.1016/j.joen.2011.02.013
JOE — Volume 37, Number 5, May 2011
O cclusal trauma is defined as an injury to any component of the masticatory system as
a result of an alteration in the occlusion that could generate an inflammatory
process in periodontal ligament and dental pulp (1). A traumatic occlusion is often
provoked by an abnormal contact of the teeth, a masticatory system dysfunction, and
prosthetic or orthodontic treatments that create occlusal interferences (2).
It has been demonstrated that occlusal trauma generates sprouts of SP-containing
fibers in pulp and periodontal ligament that could lead to a neurogenic inflammation
process (3, 4). Tissular lesions in pulp and periodontal ligament caused by traumatic
occlusion affect tissue metabolism, generating neural, vascular, and immune changes
related to an inflammatory response (5). Depending on the duration and severity of the
occlusal trauma and on previous tissue health status, damage to dental pulp might
include tissue hyalinization, pulp tissue calcification and necrosis, whereas periodontal
ligament might experience an inflammatory process, clinically identified as apical
periodontitis (1–4).
Similar to dental pulp, periodontal ligament inflammation has a neurogenic
source, induced by the release of neuropeptides from periapical tissue C-type nerve
fibers after being injured (6). Substance P (SP) is capable of triggering vasodilation,
plasma extravasation, immune system activation, chemotaxis, and recruitment and/or
regulation of inflammatory cells such as macrophages, mast cells, and lymphocytes
(7). Finally, the release of inflammatory mediators in the tissue generates vascular stasis
in the affected area (3, 8). 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 (9, 10).
SP and its neurokinin 1 (NK1) receptors have been identified both in dental pulp
(11, 12) as well as in periodontal ligament (13, 14). In dental pulp, it has been reported
that its expression increases with carious progression (15) and with restorative proce-
dures such as tooth bleaching (16), cavity preparation (17), and dentin bonding (18).
SP has also been implicated during periodontal ligament inflammatory process (13, 19,
20).
Taking into consideration the multiple anatomical and functional relationships
between dental pulp and periodontal ligament (such as apical foramen, lateral canals,
and dentinal tubules), it is reasonable to assume that this close relationship facilitates
the extension of the inflammatory process from one tissue to another (21), and conse-
Occlusal Trauma Effect on SP 627
Clinical Research
quently, dental tissues might suffer extensive damage if the stimulus is
not eliminated. Therefore, occlusal trauma might trigger a neurogenic
inflammation response in both tissues at the same time, and SP might
play an important role during this inflammatory process. The purpose
of this study was to quantify the effect of occlusal trauma experimentally
induced with occlusal interferences on SP in healthy human dental pulp
and periodontal ligament.
Materials and Methods
An experimental study was performed according to Colombian
Ministry of Health recommendations regarding ethical issues in
research involving human tissue. Written informed consent was ob-
tained from each of the 7 patients participating in the study (18 to 35
years old, healthy, not medicated, and nonsmoking human donors,
with extraction indicated in 4 premolars for orthodontic reasons).
For each patient, 2 upper and 2 lower premolars were used. All teeth
used were caries-free and restoration-free, with complete root develop-
ment determined both visually and radiographically, without signs of
periodontal disease or traumatic occlusion, and without orthodontic
forces.
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. Left upper and lower premolars were assigned to
the occlusal trauma group, and the contralateral premolars were as-
signed to the control group, for a total of 14 teeth in each group. An
occlusal interference was placed on the lower teeth in the occlusal
trauma group as follows. Articulating paper was used to mark the
contact area between the upper and lower premolars indicated for
extraction. Once established, the marked area on the lower premolar
was acid-etched with 37% phosphoric acid (SuperEtch; SDI Ltd, Bays-
water, Victoria, Australia) for 15 seconds, washed, and dried. A bonding
agent (STAE; SDI Ltd) was placed and light-cured for 15 seconds, and
finally a 1- to 2-mm block of resin (ICE; SDI Ltd) was placed over the
contact area and light-cured for 40 seconds. Articulating paper was
used again to verify that only the premolars that were going to be ex-
tracted had contact during normal occlusion as well as in lateral move-
ments. Patients were given chewing gum and indications to repeat 20
masticatory cycles for 30 seconds, followed by a 30-second rest interval,
and repeat the sequence again during a period of 30 minutes.
Experimental Procedure and Sample Collection
Teeth in the control and occlusal trauma groups were extracted 10
minutes after the 30-minute gum chewing period with conventional
methods and without excessive injury to periodontal ligament. Immedi-
ately after extraction, periodontal ligament 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. The teeth were then sectioned by using a Zekrya bur (Dentsply,
TABLE 1. SP Expression in Human Dental Pulp from Healthy Human Premolars with and without Occlusal Trauma Experimentally Induced with Occlusal
Interferences
No. of patients No. of teeth
Control group 7 14
Occlusal trauma group 7 14
Paired t test
628 Caviedes-Bucheli et al.
Tulsa, OK) in a high-speed handpiece irrigated with saline solution.
Pulp tissue was obtained by using a sterile endodontic excavator, placed
on an Eppendorf tube, snap-frozen in liquid nitrogen, and kept at
–70 C until use.
Radioimmunoassay
Dental pulp and periodontal ligament samples were defrosted
without thermal shock, dried on a filter, and individually weighed on
an analytical balance. Neuropeptides were extracted by adding 150
mL 0.5 mol/L 1 acetic acid and double-boiling in a thermostat bath
for 30 minutes in accordance with previously reported protocols
(11, 12, 16–18).
SP expression was determined by competition binding assays with
a human SP-RIA kit from Phoenix Peptide Pharmaceutical (Ref. RK-
061-05; Belmont, CA). Fifty microliters of each sample solution was
incubated in polypropylene tubes at room temperature for 20 hours
with 100 mL of primary antibody and 100 mL of different SP concentra-
tions (10 pg mL 1, –1280 pg mL 1). Then, 50 mL of 125I-SP was
added and left to incubate for another 24 hours. Bound fractions
were precipitated by the addition of 100 mL of a secondary antibody
(goat anti-rabbit immunoglobulin G serum), 100 mL of normal rabbit
serum, and 500 mL of radioimmunoassay buffer containing 1% polyeth-
ylene glycol 4000. After 2 hours of incubation at room temperature,
tubes were spun at 3000 rpm for 45 minutes at 4 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 SP
present in every sample by 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 SP concentration in picomoles per milli-
gram of tissue (pulp or periodontal ligament). Mean and standard devi-
ation values are presented for each group. Paired t tests were performed
to establish statistically significant differences between groups (P < .05).
Results
SP was found to be present in all dental pulp and periodontal liga-
ment samples. For the dental pulp analysis (Table 1), mean SP expres-
sion in the control group was 821  201 pmol SP per mg of dental pulp,
whereas the expression in the occlusal trauma group was 1190  378
pmol SP per mg of dental pulp, accounting for 45% increase. For the
periodontal ligament analysis (Table 2), mean SP expression in the
control group was 0.360  0.080 pmol SP per mg of periodontal liga-
ment, whereas the expression in the occlusal trauma group was 0.794
Tissue weight (mg) SP concentration (pmol/mg tissue)
Standard Standard
Mean deviation Mean deviation
9.54 2.49 821 201
9.25 2.71 1190 378
P = .02
JOE — Volume 37, Number 5, May 2011
 Clinical Research
TABLE 2. SP Expression in Human Periodontal Ligament from Healthy Human Premolars with and without Occlusal Trauma Experimentally Induced with Occlusal
Interferences
Tissue weight (mg) SP concentration (pmol/mg tissue)

Standard Standard
No. of patients No. of teeth Mean deviation Mean deviation
Control group 7 14 2.83 1.59 0.360 0.080
Occlusal trauma group 7 14 3.14 1.84 0.794 0.316
Paired t test P < .001

 0.316 pmol SP per mg of periodontal ligament, accounting for 120% apoptosis, leading to dental pulp degeneration such as calcification
increase. Paired t tests showed statistically significant differences and/or necrosis (30, 31).
between control and experimental groups for dental pulp (P = .02) Results also showed that there is a 120% SP increase in peri-
as well as for periodontal ligament (P < .001). odontal ligament of teeth that have been submitted to experimentally
induced occlusal trauma. This increase could be explained by the simi-
larities in the vascular, cellular, and immune responses of periodontal
Discussion ligament with dental pulp (1, 4, 6). Consequences of traumatic
Previous studies on animals have described the effect of traumatic
occlusion on periodontal ligament start with tissue compression
occlusion on dental pulp, periodontal ligament, and masticatory func-
mainly in the apical portion, causing a deformation on fibroblast cell
tion (2, 3). However, the present study was aimed to establish the levels
membrane, affecting their functions such as extracellular matrix
of SP expressed in human dental pulp and periodontal ligament as
protein synthesis (32). There are also areas where vascular stasis
a result of an occlusal trauma experimentally induced with occlusal
occurs and, therefore, localized portions of periodontal ligament
interferences.
necrosis (1, 3, 6, 19).
SP values were obtained from healthy premolars in which extrac-
Moreover, release of SP-induced inflammatory mediators generates
tion was indicated for orthodontic reasons. Local anesthetic used in all
an unbalance in the regulating mechanisms of bone and extracellular
groups of this study was 4% prilocaine without vasoconstrictor to
matrix remodeling because of increased expression of interleukin-1
prevent neuropeptide expression becoming attenuated by vasoconstric-
and prostaglandin E2 (33, 34). This could explain the pain experience
tors as previously demonstrated (22, 23). Extraction procedure was
associated with occlusal trauma, the widening of periodontal ligament,
also standardized to affect all teeth equally; it was carried out in less
tooth and bone increased resorption, and the delay in the periapical
than 5 minutes and without excessive injury to periodontal ligament.
lesion repairing process after root canal therapy (3, 4, 19, 35).
In the present study there was a 30-minute stimulation period and
It is important to be aware that the present study only reflects
a 10-minute delay after stimulation before proceeding with tooth extrac-
a model of acute inflammation. Because of the in vivo nature of the
tion. Because SP release is immediate, calcium-dependent, and of short
study and taking into consideration ethical issues, it would be difficult
term, this period of time appears to be sufficient for allowing the neuro-
to develop a similar chronic inflammation model, where it would be
peptide to be released from terminal fibers before being degraded by
necessary to leave the patients with premature contacts for a longer
endogenous peptidases (24). Other authors have speculated on the
period of time (days or weeks), which might cause pain and discomfort
possible mechanisms for the increase of extracellular neuropeptides,
to the patients.
including (1) increased synthesis of the neuropeptide in the trigeminal
Therefore, it can be suggested that pulp and periodontal ligament
ganglia, (2) increased rate of transport, (3) increased release, and (4)
response to occlusal interferences might be mediated by SP and its
decreased levels of peptidases, which would result in decreased degra-
interaction with neurokinin 1 receptors, constituting a potential expla-
dation of neuropeptides (25). More recent evidence has demonstrated
nation for the inflammatory and degenerative changes that occur in both
that mRNA transcripts are transported to peripheral terminals, suggest-
tissues. Results from the present study are in accordance with the
ing that peptide synthesis could occur directly in the peripheral termi-
hypothesis that neuropeptides contribute to the pathophysiology of
nals (26).
peripheral inflammation and that occlusal trauma generates an inflam-
Results from the present study showed that teeth that have been
matory process in dental pulp and periapical tissues that could explain
submitted to occlusal trauma experienced 45% increase in pulp SP
pain events such as symptomatic apical periodontitis (6, 13, 19). It is
levels, supporting the hypothesis that sensory nerve fibers respond to
concluded that occlusal trauma experimentally induced with occlusal
excessive masticatory forces. Mechanical irritation of dentin could affect
interferences increases SP expression in human dental pulp and
dental pulp as a result of increased fluid movement inside the dentinal
periodontal ligament, constituting a probable mechanism that could
tubules, stimulating pulp nociceptors (27). SP released from nerve
lead to an inflammatory process in the pulp and periapical tissues.
fibers can provoke a significant alteration in tissue homeostasis and
pain sensitivity by triggering the release of inflammatory mediators
(7, 28). If the occlusal interference is not eliminated, nerve fibers Acknowledgments
could become sensitized and therefore experience spontaneous The authors deny any conflicts of interest related to this study.
depolarization, enhancing pain response (1, 4, 28).
SP increase in dental pulp suggests that occlusal interferences are
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