expresion de SP en preparaciones cavitarias 2005

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The Effect of Cavity Preparation on Substance PExpression in Human Dental PulpJavier Caviedes-Bucheli, MSc,* Jose Antonio Correa-Ortız, DDS,*

Leydy Viviana Garcıa, DDS,†† Rocıo Lopez-Torres, DDS,†† Nelson Lombana, MSc,* and Hugo Roberto Munoz, 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 expressionin healthy human dental pulp. Ten pulp samples wereobtained from healthy premolars where extraction wasindicated for orthodontic reasons. Deep cavity prepa-ration (1 mm remaining dentine thickness) was per-

formed before extraction in five of these bicuspids. Allsamples were processed and 125I-SP labeled; SP wasquantified by competition assay. The results revealedSP expression in all human pulp samples. Mann-Whit-ney’s U test revealed statistically significant higherexpression in pulp from teeth where cavity preparationhad been performed compared to control values (p 0.05). These findings suggest that SP is released duringcommon dental procedures (such as cavity preparation)and its expression may have an important clinical sig-nificance in terms of experiencing inflammation andpain.

From the *Department of Graduate Studies; ††DepartmentOral Rehabilitation; and ‡‡Department Endodontics, School of Dentistry, Pontificia Universidad Javeriana, Bogota, Colombia.

Address requests for reprint to Dr. Javier Caviedes-Bucheli,School of Dentistry, Pontificia Universidad Javeriana, Cra 7 No.40-62 Building 26, Bogota, Colombia. E-mail address:[email protected].

Copyright © 2005 by the American Association of Endodontists

Dental pulp inflammation is a complex process involving a wide variety of nervousand vascular reactions which are key components of the neurogenic phenomenon

leading to pulp necrosis (1). Neuropeptides play an active role during neurogenicinflammation 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-relatedpeptide (CGRP), neurokinin A (NKA),vasoactiveintestinalpeptide (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 nerveterminals 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 alsointeracts with other inflammatory cells such as macrophages and lymphocytes, alteringits functions and inducing the expression of cyclooxigenase-2 and interleukin-10mRNAs, 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 exposureoccurs (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 knowledgecould 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 determiningthe effect of cavity preparation on SP expression in healthyhumandental pulp.

Materials and MethodsA descriptive comparative study was performed according to Colombian Ministry

of Health recommendations regarding ethical issues in research involving human tis-sue. Written informed consent wasobtained from each patientparticipating in thestudy.

Pulp samples were obtained from five different human donors (14–23 yr old) inwhomhealthy premolar extractions hadbeen indicated fororthodontic purposes.Pulpsfrom two maxillary bicuspids were used for each patient; one was assigned to thecontrol group where normal SP values were measured and the contra lateral tooth was

assigned to the experimental group where SP values were measured after performingcavity preparations. All teeth used in this study were caries- and restoration-free withcomplete root development determined both visually and radiographically.

Digital periapical radiographs (Digital Dental Systems) were taken for every toothin the experimental group, using a standardized parallel technique to allow calibratingthe measurements taken and establishing the distance between buccal cuspid andbuccal pulp horn within0.2 mm. Half a millimeter was subtracted from this distanceand recorded as being maximum cavity depth.

Teeth were anesthetized by 1.8 ml 4% prilocaine infiltration injection. Cavitieswere 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 highspeed handpiece (GENTLEforce 7000 C, KaVo, Germany) at 20 psi air pressure withabundant irrigation. The bur was used with an intermittent brushing motion until it

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reached maximum cavity depth. Extraction was accomplished 10 minlater by conventional methods.

The teeth were washed with 5.25% sodium hypochlorite after ex-traction to eliminate remains of periodontal ligament that could havecontaminated the pulp sample. The teeth were then sectioned using a Zekrya bur (Dentsply Maillefer) in a high-speed handpiece irrigatedwith saline solution. Pulp tissue was obtained by using a sterile end-odontic excavator, fixed in 4% paraformaldehyde and then kept frozenat 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 toanother tube.

One hundred microliters of each sample’s supernatant were sub-mitted to competition bindingassays with 50l 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 for1 h (Beckman) to precipitate the bound fractions. The supernatantswere decanted and pellet radioactivity was read on a Gamma Counter(Gamma Assay LS 5500 Beckman). Scatchard analysis of the bindingdata assessed the amount of SP present in every sample.

Statistical Analysis

Values are presented as SP amount in pg per ml of dental pulpsuspension. Mean and maximum/minimum values are presented foreach group. Mann-Whitney’s U test was performed for establishing sta-tistically significant differences (p 0.05) between the groups.

ResultsSP was found to be expressed in all pulp samples (Table 1). Ex-

perimental group expression was between 576.18 pg/ml and 5065.91pg/ml dental pulp suspension. Control group expression was between201.11 pg/ml and 1246.21 pg/ml. Means were 2803.05 pg/ml and664.13 pg/ml, respectively.

Mann-Whitney’s U test revealed statistically significant higher ex-pression in the pulps from teeth where cavity preparation had beenperformed compared to control values (p 0.014).

DiscussionPulpal response to restorative dentistry dependson 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 alsodepend 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 andtransient (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 effectivestimulus for releasing neuropeptides in dental pulp (20). It is alsoknown that mechanical pulp exposure alters neuropeptide levels ininflamed pulps (12, 13). Results from this study broaden knowledge

regarding SP physiology during cavity preparation, showing a signifi-cantly higher expression in pulps shortly after cavity preparation whencompared 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 individualsare more susceptible to pain and inflammation after a procedure andconsequently 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 pulpexposure. This experimental procedure was carried out following allcurrently accepted parameters for cutting dentin. A new cylindricaldiamond bur was used for each preparation to assure an effective cut avoiding to exert excessive pressure on the tooth (21, 22); water-spray coolingeffectiveness wasassured with a four-hole irrigationhand-piece(23, 24); air pressure was set at 20 psi to achieve a rotational speed of the burin contact with the tooth of 220,000 to 260,000 rpm as stated by the manufacturer (KaVo, Germany) and intermittent brushing motionwas used to reduce frictional heat (25).

Local anesthetic used in this study was 4% prilocaine without va-soconstrictor to prevent neuropeptide expression becoming attenuatedby Alpha-adrenergic agonists (e.g. vasoconstrictors) as stated by otherauthors (26, 27). There was a 10-min delay after preparing the cavity before proceeding with tooth extraction. It has been shown that thisperiod of time appears to be sufficient for allowing the neuropeptide tobe released from terminal fibers (26).

The methods used in this study could be useful in establishing theexpression of other neuropeptides after cavity preparations, especially CGRPthathasbeenshowntobehighlyactiveinpulpalinflammationandmay modulate the inflammatory response (28).

It should be noted that this study was carried out on caries- andrestoration-free teeth; it is thus important to be aware of these findings’limitations. It has been demonstrated that caries-affected teeth showeda significant increase in SP, CGRP, VIP, and NPY expression with cariesprogression (29). However, the present evidence could have biological

and clinical importance in connection with future research regardingnociception, inflammation, and healing process following restorativeprocedures.

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4. Wakisaka S. Neuropeptides in the dental pulp: distribution, origins and correlations.J Endod 1990;16:67–9.

5. WakisakaS, AkaiM. Immunohistochemicalobservationon neuropeptidesaroundtheblood vessel in feline dental pulp. J Endod 1989;15:413– 6.

TABLE 1. SP expression on healthy human dental pulp from teeth with andwithout cavity preparation

PatientNormal SP

Levels*SP Expression afterCavity Preparation*

(control tooth) (experimental tooth)1 440.81 5017.092 407.18 5065.913 201.11 576.184 1025.33 1327.15

5 1246.21 2028.94Mean 664.12 2803.05p value 0.014†

*Values are given in pg SP per ml dental pulp suspension.

†Differences between groups were statistically significant.

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