Clinical Research
The Effect of Cavity Preparation on Substance P
Expression in Human Dental Pulp
Javier Caviedes-Bucheli, MSc,* José Antonio Correa-Ortı́z, DDS,*
Leydy Viviana Garcı́a, DDS,†† Rocı́o López-Torres, DDS,†† Nelson Lombana, MSc,* and
Hugo Roberto Muñoz, MSc‡‡
Abstract
Substance P (SP) plays an important role during neu-
rogenic inflammation of dental pulp. The purpose of
this study was to use a radioimmunoassay for deter-
mining the effect of cavity preparation on SP expression
in healthy human dental pulp. Ten pulp samples were
obtained from healthy premolars where extraction was
indicated for orthodontic reasons. Deep cavity prepa-
ration (⬍1 mm remaining dentine thickness) was per-
formed before extraction in five of these bicuspids. All
samples were processed and 125I-SP labeled; SP was
quantified by competition assay. The results revealed
SP expression in all human pulp samples. Mann-Whit-
ney’s U test revealed statistically significant higher
expression in pulp from teeth where cavity preparation
had been performed compared to control values (p ⬍
0.05). These findings suggest that SP is released during
common dental procedures (such as cavity preparation)
and its expression may have an important clinical sig-
nificance in terms of experiencing inflammation and
pain.
From the *Department of Graduate Studies; ††Department
Oral Rehabilitation; and ‡‡Department Endodontics, School of
Dentistry, Pontificia Universidad Javeriana, Bogotá, Colombia.
Address requests for reprint to Dr. Javier Caviedes-Bucheli,
School of Dentistry, Pontificia Universidad Javeriana, Cra 7 No.
40-62 Building 26, Bogotá, Colombia. E-mail address:
javiercaviedes@cable.net.co.
Copyright © 2005 by the American Association of
Endodontists
JOE — Volume 31, Number 12, December 2005
D ental pulp inflammation is a complex process involving a wide variety of nervous
and vascular reactions which are key components of the neurogenic phenomenon
leading to pulp necrosis (1). Neuropeptides play an active role during neurogenic
inflammation of the pulp, controlling its blood flow and regulating later stages of
inflammation and repair process (2, 3). These neuropeptides include substance P
(SP), calcitonin gene-related peptide (CGRP), neurokinin A (NKA), vasoactive intestinal
peptide (VIP), and neuropeptide Y (4).
SP is produced in trigeminal cell bodies and transported via axonal flow to nerve
terminals in the pulp where it is stored with other neuropeptides (5). These nerve
terminals are mainly C-type fibers that are closely related to pulp microcirculation,
neuropeptides being released when terminals are stimulated (6). It has been demon-
strated that SP interacts with mastocytes, inducing the release of histamine and thereby
causing elevated vascular permeability and increased blood pressure in tissue. It also
interacts with other inflammatory cells such as macrophages and lymphocytes, altering
its functions and inducing the expression of cyclooxigenase-2 and interleukin-10
mRNAs, having a direct effect on pulp tissue (7–10) and periapical granulomas (11).
A body of experimental evidence supports the fact that SP expression significantly
increases in the pulp when acute irreversible pulpitis or mechanical pulp exposure
occurs (1, 6, 12–14). However, little is known about how much SP is released when a
tooth becomes injured by cavity preparation without pulp exposure. This knowledge
could be useful for correlating this neuropeptide’s behavior when routine restorative
procedures are carried out. The purpose of this study was thus to use a radioimmuno-
assay for determining the effect of cavity preparation on SP expression in healthy human
dental pulp.
Materials and Methods
A descriptive comparative study was performed according to Colombian Ministry
of Health recommendations regarding ethical issues in research involving human tis-
sue. Written informed consent was obtained from each patient participating in the study.
Pulp samples were obtained from five different human donors (14 –23 yr old) in
whom healthy premolar extractions had been indicated for orthodontic purposes. Pulps
from two maxillary bicuspids were used for each patient; one was assigned to the
control group where normal SP values were measured and the contra lateral tooth was
assigned to the experimental group where SP values were measured after performing
cavity preparations. All teeth used in this study were caries- and restoration-free with
complete root development determined both visually and radiographically.
Digital periapical radiographs (Digital Dental Systems) were taken for every tooth
in the experimental group, using a standardized parallel technique to allow calibrating
the measurements taken and establishing the distance between buccal cuspid and
buccal pulp horn within ⫾0.2 mm. Half a millimeter was subtracted from this distance
and recorded as being maximum cavity depth.
Teeth were anesthetized by 1.8 ml 4% prilocaine infiltration injection. Cavities
were prepared 5 min later in each tooth from the experimental group with a new
cylindrical diamond bur No. 515.7C (Two Striper Diamonds, Premier, USA) in a high
speed handpiece (GENTLEforce 7000 C, KaVo, Germany) at 20 psi air pressure with
abundant irrigation. The bur was used with an intermittent brushing motion until it
SP Expression in Cavity Preparation 857
Clinical Research
reached maximum cavity depth. Extraction was accomplished 10 min
later by conventional methods.
The teeth were washed with 5.25% sodium hypochlorite after ex-
traction to eliminate remains of periodontal ligament that could have
contaminated the pulp sample. The teeth were then sectioned using a
Zekrya bur (Dentsply Maillefer) in a high-speed handpiece irrigated
with saline solution. Pulp tissue was obtained by using a sterile end-
odontic excavator, fixed in 4% paraformaldehyde and then kept frozen
at ⫺70°C until use.
Radioimmunoanalysis (RIA)
Pulp samples were ultrasonically disaggregated (Ultrasonic Pro-
cessor S-2028-130, ISC BioExpress, Kaysville, UT) for their homogeni-
zation; 250 ␮l acetic acid was added and double-boiled for 10 min.
Disaggregated tissue was spun at 3500 rpm for 45 min (GS-6KR Cen-
trifuge, Beckman, Fullerton, CA) and supernatants were transferred to
another tube.
One hundred microliters of each sample’s supernatant were sub-
mitted to competition binding assays with 50 ␮l 125I-SP (Amersham Ref.
IM57, Piscataway NJ), 50 ␮l 1:100 anti-SP solution (Ref. S-1542,
Sigma, St. Louis, MO), 50 ␮l different unlabeled SP (Sigma S-6883)
concentrations and 500 ␮l polyethylenglycol (Sigma P-2139).
After 2 h incubation, the suspensions were spun at 5000 rpm for
1 h (Beckman) to precipitate the bound fractions. The supernatants
were decanted and pellet radioactivity was read on a Gamma Counter
(Gamma Assay LS 5500 Beckman). Scatchard analysis of the binding
data assessed the amount of SP present in every sample.
Statistical Analysis
Values are presented as SP amount in pg per ml of dental pulp
suspension. Mean and maximum/minimum values are presented for
each group. Mann-Whitney’s U test was performed for establishing sta-
tistically significant differences (p ⬍ 0.05) between the groups.
Results
SP was found to be expressed in all pulp samples (Table 1). Ex-
perimental group expression was between 576.18 pg/ml and 5065.91
pg/ml dental pulp suspension. Control group expression was between
201.11 pg/ml and 1246.21 pg/ml. Means were 2803.05 pg/ml and
664.13 pg/ml, respectively.
Mann-Whitney’s U test revealed statistically significant higher ex-
pression in the pulps from teeth where cavity preparation had been
performed compared to control values (p ⫽ 0.014).
Discussion
Pulpal response to restorative dentistry depends on several factors
including thermal and mechanical irritation, damage to odontoblastic
processes, thickness of remaining dentin and dental materials biocom-
patibility (15, 16). Effects of cavity preparation on pulp tissue also
depend on other factors such as wear and design of the bur used,
rotational speed and torque, the amount of force applied to the bur,
cutting time, operative technique, and the cooling efficiency of the irri-
gant (17, 18). However, a healthy pulp is able to defend itself and most
of the effects of restorative procedures on the pulp are minor and
transient (19).
Limited evidence has been presented to date regarding the behav-
ior of neuropeptide release during common restorative procedures. It
has been shown that high-speed drilling in rat’s teeth is an effective
stimulus for releasing neuropeptides in dental pulp (20). It is also
known that mechanical pulp exposure alters neuropeptide levels in
inflamed pulps (12, 13). Results from this study broaden knowledge
858 Caviedes-Bucheli et al.
TABLE 1. SP expression on healthy human dental pulp from teeth with and
without cavity preparation
Normal SP SP Expression after
Patient
Levels* Cavity Preparation*
(control tooth) (experimental tooth)
1 440.81 5017.09
2 407.18 5065.91
3 201.11 576.18
4 1025.33 1327.15
5 1246.21 2028.94
Mean 664.12 2803.05
p value 0.014†
*Values are given in pg SP per ml dental pulp suspension.
†Differences between groups were statistically significant.
regarding SP physiology during cavity preparation, showing a signifi-
cantly higher expression in pulps shortly after cavity preparation when
compared to normal neuropeptide levels.
It is interesting to notice that control SP values showed great vari-
ation between patients. This could be the reason why some individuals
are more susceptible to pain and inflammation after a procedure and
consequently tend to require root canal therapy more than others. How-
ever, future research is needed to ascertain this.
SP release was induced by a deep cavity preparation without pulp
exposure. This experimental procedure was carried out following all
currently accepted parameters for cutting dentin. A new cylindrical
diamond bur was used for each preparation to assure an effective cut
avoiding to exert excessive pressure on the tooth (21, 22); water-spray
cooling effectiveness was assured with a four-hole irrigation hand-piece
(23, 24); air pressure was set at 20 psi to achieve a rotational speed of
the bur in contact with the tooth of 220,000 to 260,000 rpm as stated by
the manufacturer (KaVo, Germany) and intermittent brushing motion
was used to reduce frictional heat (25).
Local anesthetic used in this study was 4% prilocaine without va-
soconstrictor to prevent neuropeptide expression becoming attenuated
by Alpha-adrenergic agonists (e.g. vasoconstrictors) as stated by other
authors (26, 27). There was a 10-min delay after preparing the cavity
before proceeding with tooth extraction. It has been shown that this
period of time appears to be sufficient for allowing the neuropeptide to
be released from terminal fibers (26).
The methods used in this study could be useful in establishing the
expression of other neuropeptides after cavity preparations, especially
CGRP that has been shown to be highly active in pulpal inflammation and
may modulate the inflammatory response (28).
It should be noted that this study was carried out on caries- and
restoration-free teeth; it is thus important to be aware of these findings’
limitations. It has been demonstrated that caries-affected teeth showed
a significant increase in SP, CGRP, VIP, and NPY expression with caries
progression (29). However, the present evidence could have biological
and clinical importance in connection with future research regarding
nociception, inflammation, and healing process following restorative
procedures.
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