The Effect of Cavity Preparation on Substance P Expression in Human Dental Pulp

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The Effect of Cavity Preparation on Substance P Expression in Human Dental Pulp
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  The Effect of Cavity Preparation on Substance PExpression in Human Dental Pulp   Javier Caviedes-Bucheli, MSc,* Jose´ Antonio Correa-Ortı´z, DDS,*  Leydy Viviana Garcı´a, DDS, ††   Rocı´o Lo´pez-Torres, DDS, ††   Nelson Lombana, MSc,* and  Hugo Roberto Mun˜oz, 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  125 I-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: javiercaviedes@cable.net.co.Copyright © 2005 by the American Association of Endodontists D ental pulp inflammation is a complex process involving a wide variety of nervousand vascular reactions which are key components of the neurogenic phenomenonleading 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),calcitoningene-relatedpeptide(CGRP),neurokininA(NKA),vasoactiveintestinalpeptide (VIP), and neuropeptide Y (4).SP is produced in trigeminal cell bodies and transported via axonal flow to nerveterminals 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 restorativeprocedures are carried out. The purpose of this study was thus to use a radioimmuno-assayfordeterminingtheeffectofcavitypreparationonSPexpressioninhealthyhumandental 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.Writteninformedconsentwasobtainedfromeachpatientparticipatinginthestudy.Pulp samples were obtained from five different human donors (14–23 yr old) in whomhealthypremolarextractionshadbeenindicatedfororthodonticpurposes.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 wasassigned 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 within  0.2 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. 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 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  Clinical Research   JOE —   Volume 31, Number 12, December 2005 SP Expression in Cavity Preparation  857  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 irrigated with 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-mittedtocompetitionbindingassayswith50  l 125 I-SP(AmershamRef.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 supernatants were 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. Results SP was found to be expressed in all pulp samples (Table 1). Ex-perimental group expression was between 576.18 pg/ml and 5065.91pg/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). Discussion Pulpalresponsetorestorativedentistrydependsonseveralfactorsincluding thermal and mechanical irritation, damage to odontoblasticprocesses, 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 knowledgeregarding 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 andconsequentlytendtorequirerootcanaltherapymorethanothers.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 coolingeffectivenesswasassuredwithafour-holeirrigationhand-piece(23, 24); air pressure was set at 20 psi to achieve a rotational speed of theburincontactwiththetoothof220,000to260,000rpmasstatedby 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-soconstrictortopreventneuropeptideexpressionbecomingattenuatedby 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,thepresentevidencecouldhavebiologicaland clinical importance in connection with future research regardingnociception, inflammation, and healing process following restorativeprocedures. References 1. Kim S. Neurovascular interactions in the dental pulp in health and inflammation. J Endod 1990;16:48–53.2. OlgartL.Neuralcontrolofpulpalbloodflow.CritRevOralBiolMed1996;7:159–71.3. Olgart L. Neurogenic components of pulp inflammation. In: Shimono M, Maeda T,SudaH,TakahashiK,eds.Dentine/pulpcomplex.Tokyo:QuintessenceBooks,1996:169–75.4. Wakisaka S. Neuropeptides in the dental pulp: distribution, srcins and correlations. J Endod 1990;16:67–9.5. WakisakaS,AkaiM.Immunohistochemicalobservationonneuropeptidesaroundtheblood vessel in feline dental pulp. J Endod 1989;15:413–6. TABLE 1.  SP expression on healthy human dental pulp from teeth with and without cavity preparation Patient Normal SPLevels*SP Expression afterCavity Preparation* (control tooth) (experimental tooth)1 440.81 5017.092 407.18 5065.913 201.11 576.184 1025.33 1327.155 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. Clinical Research 858  Caviedes-Bucheli et al.  JOE   — Volume 31, Number 12, December 2005  6. AwawdehL,LundyFT,ShawC,LameyPJ,LindenGJ,KennedyJG.Quantitativeanalysisof substance P, neurokinin A and calcitonin gene-related peptide in pulp tissue frompainful and healthy human teeth. Int Endod J 2002;35:30–6.7. Hargreaves KM, Swift JQ, Roszkowski MT, Bowles W, Garry MG, Jackson DL. Phar-macology of peripheral neuropeptide and inflammatory mediator release. Oral SurgOral Med Oral Pathol 1994;78:503–10.8. Patel T, Park SH, Lin LM, Chiapelli F, Huang GT. Substance P induces interleukin-8secretion from human dental pulp cells. Oral Surg Oral Med Oral Pathol Oral RadiolEndod 2003;96:478–85.9. ParkSH,HsiaoGY,HuangGT.RoleofsubstancePandcalcitoningene-relatedpeptidein the regulation of interleukin-8 and monocyte chemotactic protein-1 expression inhuman dental pulp. Int Endod J 2004;37:185–92.10. Tokuda M, Miyamoto R, Nagaoka S, Torii M. Substance P enhances expression of lipopolysaccharide-induced inflammatory factors in dental pulp cells. J Endod 2004;30:770–4.11. Tuncer LI, Alacam T, Oral B. Substance P expression is elevated in inflamed humanperiradicular tissue. J Endod 2004;30:329–32.12. Buck S, Reese K, Hargreaves KM. Pulpal exposure alters neuropeptide levels ininflamed dental pulp and trigeminal ganglia: evaluation of axonal transport. J Endod1999;25:718–21.13. Bowles WR, Withrow JC, Lepinski AM, Hargreaves KM. Tissue levels of immunoreac-tive substance P are increased in patients with irreversible pulpitis. J Endod 2003;29:265–7.14. RoddHD,BoissonadeFM.SubstancePexpressioninhumantoothpulpinrelationtocaries and pain experience. Eur J Oral Sci 2000;108:467–74.15. AboutI,MurrayPE,FranquinJC,RemusatM,SmithAJ.Theeffectofcavityrestoration variables on odontoblast cell number and dental repair. J Dent 2001;29:109–17.16. MurrayPE,AboutI,LumleyPJ,FranquinJC,RemusatM,SmithAJ.Humanodontoblast cell numbers after dental injury. J Dent 2000;28:277–85.17. Lee SJ, Walton RE, Osborne JW. Pulp response to bases and cavity depths. Am J Dent 1992;5:64–8.18. Hatton JF, Holtzmann DJ, Ferrillo PJ, Stewart GP. Effect of handpiece pressure andspeed on intrapulpal temperature rise. Am J Dent 1994;7:108–10.19. Murray PE, About I, Lumley PJ, Smith G, Franquin JC, Smith AJ. Postoperative pulpaland repair responses. J Am Dent Assoc 2000;131:321–9.20. Takamori K. A histopathological and immunohistochemical study of dental pulp andpulpal nerve fibers in rats after the cavity preparation using Er:YAG laser. J Endod2000;26:95–9.21. Ottl P, Lauer CH. Temperature response in the pulpar chamber during ultrahigh-speed tooth preparation with diamond burs on different grit. J Prosthet Dent 1998;80:12–9.22. Galindo DF, Ercoli C, Funkenbusch PD, et al. Tooth preparation: a study on the effect of different variables and a comparison between conventional and channeled dia-mond burs. J Prosthodont 2004;13:3–16.23. LauerCH,KraftE,RothlaufW,ZwingersT.Effectsofthetemperatureofcoolingwaterduring high-speed tooth preparation. J Prosthet Dent 1990;63:407–14.24. LockardMW.Aretrospectivestudyofpulparresponseinvitaladultteethpreparedforcomplete coverage restorations at ultrahigh speed using only air cooling. J Prosthet Dent 2002;88:473–8.25. OzturkB,UsumezA,OzturkAN,OzerF.Invitroassessmentoftemperaturechangeinthe pulp chamber during cavity preparation. J Prosthet Dent 2004;91:436–40.26. HargreavesKM,JacksonDL,BowlesWR.Adrenergicregulationofcapsaicin-sensitiveneurons in dental pulp. J Endod 2003;29:397–9.27. Pertl C, Amann R, Odell E, Robinson PD, Kim S. Effects of local anesthesia on Sub-stance P and CGRP content of the human dental pulp. J Endod 1997;23:416–8.28. Caviedes-Bucheli J, Camargo-Beltra´n C, Go´mez-la-Rotta AM, Moreno SC, Abello GC,Gonza´lez-Escobar JM. Expression of calcitonin gene-related peptide (CGRP) in irre- versible acute pulpitis. J Endod 2004;30:201–4.29. Rodd HD, Boissonade FM. Comparative immunohistochemical analysis of the pepti-dergic innervation of human primary and permanent tooth pulp. Arch Oral Biol2002;47:375–85. Clinical Research   JOE —   Volume 31, Number 12, December 2005 SP Expression in Cavity Preparation  859
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