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Clinical Research
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he Effect of Tooth Bleaching on Substance P Expressionn Human Dental Pulpavier Caviedes-Bucheli, DDS, MSc,* Germán Ariza-García, DDS,†
ilvia Restrepo-Méndez, DDS,* Nestor Ríos-Osorio, DDS,* Nelson Lombana, PhD,‡ andugo Roberto Muñoz, DDS, MA§
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bstracthe purpose of this study was to quantify the effect ofooth bleaching on substance P (SP) expression in healthyuman dental pulp. Forty pulp samples were obtainedrom healthy premolars in which extraction was indicatedor orthodontic reasons. Thirty of these premolars weressigned into three different tooth-bleaching protocols:roup 1 (n � 10): Opalescence Xtra Boost (Ultradentroducts, South Jordan, UT) (38% H2O2) for 15 minutes;roup 2 (n � 10): Lase Peroxide (DMC, Brazil) (35% H2O2)ctivated with infrared laser diode (Biolux; BioArt, Brazil)or 3 minutes, and group 3 (n � 10): Zoom! Whiteningystem (Discuss Dental, Culver City, CA) (25% H2O2) lightctivated for 20 minutes. The remaining 10 healthy pre-olars serve as a control group. Teeth were anesthetized
mmediately after bleaching and were extracted 10 min-tes later. All pulp samples were processed and SP waseasured by radioimmunoassay. Greater SP expressionas found in the Zoom! Whitening System, followed by
he Lase Peroxide group, Opalescence Xtra Boost, and theower SP values were for the control group. Analysis ofariance showed statistically significant differences be-ween groups (p � 0.0001). Tukey HSD post hoc testshowed significant differences in the light (p � 0.01) andaser (p � 0.05) activated bleaching systems when com-ared with control values. It can be concluded that light-nd laser-activated tooth-bleaching systems increase SPxpression in human dental pulp significantly higher thanormal values. (J Endod 2008;34:1462–1465)
ey Wordsuman dental pulp, neuropeptide, substance P, toothleaching
From the Departments of *Endodontics and †Oral Reha-ilitation, School of Dentistry, Pontificia Universidad Javeriana,nd the ‡Department of Chemistry, Universidad de los Andes,ogotá, Colombia; and §Department of Endodontics, School ofentistry, Universidad de San Carlos de Guatemala, Guate-ala.
Address requests for reprints to Javier Caviedes-Bucheli,DS, MSc, School of Dentistry, Pontificia Universidad Javeri-na, Cra 7 No. 40-62 Building 26, Bogotá, Colombia. E-mailddress: [email protected]/$0 - see front matter
Copyright © 2008 American Association of Endodontists.oi:10.1016/j.joen.2008.09.013
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462 Caviedes-Bucheli et al.
ental pulp inflammation is a complex process involving a wide variety of neuronaland vascular reactions that are key components of the neurogenic inflammation
hat could lead to pulp necrosis (1). Neuropeptides are the major protagonists of theeuronal component and are present on the sensitive afferent neurons from the trigem-
nal ganglia and on sympathetic fibers from the cervical ganglia (2, 3). Substance P (SP)s, among others, the most important neuropeptide. Its primary function is to induceasodilation, increasing pulpal blood flow allowing rapid and large arrival of inflam-atory cells and mediators release into the inflammatory site (4).
Pulp tissue undergoes several alterations during neurogenic inflammation, includ-ng hyperalgesia or sensitization of nerve fibers causing a reduction of pain threshold,ncreased inflammatory response because of the arrival of vasoactive substances, andxtravasation of fluids and plasmatic proteins to the interstitial tissue, thereby increasingulp pressure on the injury site. These alterations could have permanent consequences
n a low compliance environment, such as dental pulp (5).Contemporary dentistry is strongly influenced by esthetics, where tooth bleaching
as become a usually requested and/or offered clinical procedure although somearmful effects over oral tissues, including dental pulp, have been associated with this
reatment (6, 7). Chemicals commonly used for tooth bleaching usually contain someorm of hydrogen peroxide (H2O2) at different concentrations. H2O2 is an oxidant agenthat, with the aid of some chemical or physical activators (ie, enzymes, light or heat),issociates in perhydroxyl (HO2�) and oxygen (O�) radicals. These free radicals,nown as reactive oxygen species, have low molecular weight and are able to denatu-alize proteins, penetrate enamel, and diffuse through the organic matrix of dentin toxert its bleaching effect (8, 9).
Most of the current in-office tooth-bleaching systems use light and/or heat as thectivator source to enhance bleaching efficacy by accelerating the oxygen dissociationate, thus reducing the time needed to whiten teeth. It has been shown that by each 10°Cncrease in temperature the rate of chemical reactions within the bleaching agentuplicates, generating greater dissociation and release of free radicals, which will reactore rapidly with pigmented molecules of the tooth (10-12). However, it also has been
eported that a 5°C increase in pulp temperature could have irreversible consequenceso dental pulp (13). Different light-energy sources have been used to activate bleachinggents, including halogen-curing lights, ultraviolet (UV) and infrared lamps, and lasersCO2, argon, and more recently laser diode) (14).
During bleaching, a reduction/oxidation reaction occurs also known as redoxeaction. The oxidant agent, H2O2, dissociates into free radicals with unpaired electronsnd gets reduced by giving these electrons, and, consequently, the substances beingleached get oxidized by accepting the electrons. This results in a molecular rupturend in a change in the energy absorption at the molecular level within the pigmentedental organic matrix, forming simpler and smaller molecules that reflect light in aifferent way, creating a successful bleaching effect (15, 16). However, free radicalseleased during bleaching are highly reactive and have been associated with severalhysiologic and pathological events, including proteins, lipids, and nucleic acids deg-adation, leading to tissue aging and other degenerative processes (9, 15). The pres-nce of dentinal tubules within the tooth architecture could allow these free radicals to
each the pulp tissue and generate these adverse effects (13, 17).JOE — Volume 34, Number 12, December 2008
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It has been shown that, in cultured fibroblasts, H2O2 is toxic toells even in lower concentrations like 10 �mol/L (0.34 mg/L), inhib-ting cell proliferation by DNA chain breakage (18). The diffusion ofifferent H2O2 concentrations through dentin has also been evaluated,howing that only 15 minutes are needed for H2O2 to diffuse through a.5-mm width of dentin, reaching concentrations capable of generatingiological damage in fibroblasts cultures (19).
Tooth-bleaching procedures often generate some type of clinicalymptoms, from dentin hypersensitivity to reversible or irreversible in-lammatory processes, in which SP could play an important role in theevelopment of these conditions (20). Up to date, tooth-bleaching ef-ects on pulp tissue have been studied by histological methods; however,ittle is known about neuropeptide release after this procedure. Thisnowledge could be useful for assessing SP behavior when routinelinical procedures are performed and, consequently, contribute tolinician’s decision making in order to minimize pulp tissue injury.herefore, the purpose of this study was to determine the effect ofifferent tooth-bleaching protocols on SP expression in healthy humanental pulp.
Materials and MethodsA descriptive comparative study was performed according to Co-
ombian Ministry of Health recommendations regarding ethical issuesn research involving human tissue. Written informed consent was ob-ained from each patient participating in the study. Forty pulp samplesere obtained from healthy, nonsmoking human donors (18-27 yearsld) in whom healthy premolar extractions had been indicated forrthodontic purposes. All teeth used in this study were caries and res-oration free with complete root development determined both visuallynd radiographically and without signs of periodontal disease.
Teeth were divided into four groups containing five upper and fiveower premolars each: (1) control group: 10 healthy pulps in whichormal SP values were measured, (2) experimental group 1: 10 healthyremolars in which a 15-minute 38% H2O2 (Opalescence Xtra Boost;ltradent Products, South Jordan, UT) bleaching procedure was per-
ormed, (3) experimental group 2: 10 healthy premolars in which a5% H2O2 (Lase Peroxide, DMC, São Paulo, Brazil) activated with an
nfrared laser diode (Biolux, BioArt, São Paulo, Brazil) bleaching pro-edure was performed for 3 minutes, and (4) experimental group 3: 10ealthy premolars in which a 20-minute light-activated 25% H2O2Zoom! Whitening System; Discuss Dental, Culver City, CA) bleachingrocedure was performed. All tooth-whitening systems were used fol-owing manufacturer’s instructions.
ample CollectionTeeth were anesthetized immediately after bleaching by 1.8 mL of
% prilocaine without vasoconstrictor infiltration injection for upperremolars and by inferior alveolar nerve block injection for lower pre-olars. Ten minutes later, they were extracted by conventional meth-
ABLE 1. Pulpal SP Expression after Different Tooth-bleaching Protocols in Hea
N
Control group† (normal SP values) 10Experimental group 1† (Opalescence Xtra Boost) 10Experimental group 2‡ (Lase Peroxide and Biolux) 10Experimental group 3 (Zoom! Whitening System) 10
P, Substance P.
OTE. ANOVA test showed statistically significant differences between groups (p � 0.0001).
Values are presented as SP concentration in picomolars per milligrams of dental pulp tissue.
Tukey HSD post hoc test showed statistically significant differences with experimental group 2 (p �
Tukey HSD post hoc test also showed a statistically significant difference with experimental group 3 (p � 0
OE — Volume 34, Number 12, December 2008
ds. For the control group, extraction was also performed 10 minutesfter anesthetic application.
All teeth were washed with 5.25% sodium hypochlorite after ex-raction to eliminate remnants of periodontal ligament that could con-aminate the pulp sample. The teeth were then sectioned using a Zekryaur (Dentsply, Tulsa, OK) in a high-speed hand piece irrigated withaline solution. Pulp tissue was obtained by using a sterile endodonticxcavator, placed on an Eppendorf tube, snap frozen in liquid nitrogen,nd kept at �70°C until use.
adioimmunoassayPulp tissue samples were defrosted without thermal shock, dried
n a filter, and weighed on an analytic balance. Neuropeptides werextracted by adding 150 �L of 0.5 mol/L acetic acid and double boiledn a thermostat bath for 30 minutes.
SP expression was determined by competition binding assays us-ng a human SP radioimmunoassay kit from Phoenix Peptide Pharma-eutical (Ref. RK-061-05, Belmont, CA). The lowest detection limit ofhis kit is 55 pmol SP/mL. Fifty microliters of each sample solution werencubated in polypropylene tubes at room temperature for 20 hoursith 100 �L of primary antibody (1:100 rabbit anti-SP serum solution)nd 100 �L of different SP concentrations (10 pg/mL-1,280 pg/mL).hen, 50 �L of 125I-SP were added and left to incubate for another 24ours.
Bound fractions were precipitated by the addition of 100 �L of aecondary antibody (goat antirabbit immunoglobulin G serum), 100 �Lf normal rabbit serum, and 500 �L of radioimmunoassay buffer con-aining 1% polyethylenglycol 4000. After 2 hours of incubation at roomemperature, tubes were spun at 3,000 rpm for 45 minutes at 4°C. Theupernatants were decanted, and pellet radioactivity was read on aamma Counter (Gamma Assay LS 5500; Beckman, Fullerton, CA).tandard curves of authentic peptide were made in buffers identical tohe tissue extracts. Finally, Scatchard analysis of the binding data as-essed the amount of SP present in every sample.
tatistical AnalysisValues are presented as SP concentration in picomolars per mil-
igrams of dental pulp tissue. Mean, standard deviation, and maximum/inimum values are presented for each group. Analysis of variance
ANOVA) test was performed to establish statistically significant differ-nces between groups (p � 0.05). Tukey HSD post hoc comparisonsere also performed.
ResultsSP was found to be expressed in all pulp samples (Table 1). High-
st SP levels were observed in experimental group III (Zoom! Whiteningystem). The mean expression for this group was 1649.52 � 341.97mol SP/mg pulp tissue followed by experimental group II (Lase Per-xide activated with infrared laser diode) with a mean SP expression of
uman Premolars
an* Standard Deviation Minimum Maximum
6.94 62.85 678.16 856.320.23 141.71 578.64 1092.834.66 155.55 793.31 1321.209.52 341.97 1297.73 2107.92
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Tooth-bleaching Effect on SP 1463
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054.66 � 155.55 pmol SP/mg pulp tissue. The mean expression forhe experimental group 1 (Opalescence Xtra Boost) was 760.23 �41.71 pmol SP/mg pulp tissue. The lowest SP levels were observed inhe control group samples (healthy pulps without treatment) with a
ean expression of 756.94 � 62.85 pmol SP/mg pulp tissue. TheNOVA test showed statistically significant differences between groupsp � 0.0001). Tukey HSD post hoc tests showed significant statisticalifferences between the control group and experimental groups 2 (p �.05) and 3 (p � 0.01). The difference between experimental groupsand 3 was also statistically significant (p � 0.01). The difference
etween the control group and experimental group 1 was not statisti-ally significant.
DiscussionTooth bleaching is one of the most common procedures currently
erformed in contemporary dentistry. However, some controversy stillxists on the safety of this procedure, showing numerous articles thatefend bleaching as being innocuous to dental tissues, and, conversely,here is evidence in the literature that chemicals used for tooth bleachingave deleterious effects on pulpodentin complex (6, 7, 12, 19, 21–24).
Enamel and dentin permeability allows these low–molecular-eight radicals to penetrate into dental tissues, becoming a potentialazard to the pulp (9). However, adverse effects of tooth bleaching haveot been studied from a neurovascular approach, and neuropeptideelease in response to this procedure has not been well elucidated. In aow compliance environment, this kind of aggression could generate annflammatory response and induce pulp cells (including fibroblasts and
esenchymal undifferentiated cells) to undergo apoptosis, thereforeowering tissue ability to defend itself from future irritants (13).
Furthermore, H2O2 penetration into the pulp tissue could generateentinal sensitivity after treatment (20); current evidence suggests thatissue exposure to SP may contribute indirectly to the development ofyperalgesia. The inflammatory process generated after tooth bleachingsually last for approximately 2 weeks. During this time, proinflamma-ory cytokine release continues, which, in some cases, could perpetuateP release for longer periods of time and therefore cause posttreatmentymptoms in some cases (25, 26).
Basal SP values were obtained from healthy premolars in whichxtraction was indicated for orthodontics reasons. Local anestheticsed in all groups of this study was 4% prilocaine without vasoconstric-or to prevent neuropeptide expression becoming attenuated by alpha-drenergic agonists (ie, vasoconstrictors) as stated by previous authors27, 28). There was a 10-minute delay after bleaching procedure be-ore proceeding with tooth extraction. Because SP release is immediate,alcium dependent, and of short-term, this period of time appears to beufficient for allowing the neuropeptide to be released from terminalibers before being degraded by endogenous peptidases (29). Otheruthors (25, 30) have speculated on the possible mechanisms for thencrease of extracellular SP, including (1) increased synthesis of theeuropeptide in the trigeminal ganglia; (2) increased rate of transport;3) increased release; and (4) decreased levels of peptidases, whichould result in decreased degradation of SP. More recent evidence has
hown that messenger RNA transcripts are transported to peripheralerminals, suggesting that peptide synthesis could occur directly in theeripheral terminals (31). However, in the present study, it is assumedhat increased SP levels are more related to increased release because0 minutes would not be enough for the other hypothesis to take place.
It is important to point out that different H2O2 concentrations andifferent application times were used in the present study, and these areariables that could have influenced the results presented because it is
ssumed that adverse effects to the pulp are proportional to the con- i464 Caviedes-Bucheli et al.
entration of the bleaching agent and the time that the tooth is beingxposed to it (14, 16, 24). However, all the bleaching systems testedere used strictly following manufacturer’s instructions in order toetter simulate normal clinical conditions.
Interestingly, data from the present study showed that tooth-leaching systems using 25% and 35% H2O2 with light/heat (UV lightnd infrared laser diode) activation resulted in significantly increasedP levels when compared with basal levels and 38% H2O2 bleachingithout light/heat activation. These results confirm that light/heat appli-ation over whitening gels generates an increased reaction of H2O2 withn accelerated release of free radicals with higher kinetic energy, whichlinically traduces into a faster whitening effect (19). On the other hand,8% H2O2 tooth bleaching without light and/or heat activation does notncrease significantly the SP expression compared with control levels,uggesting that this tooth-bleaching protocol has the ability to generatewhitening effect without increasing basal SP levels. These results coulde explained by the diversity of self-defense mechanisms of the pulpo-entin complex against free radicals (32).
The first line of defense is enamel, which acts as a mechanicalarrier against external irritants (33). However, because of the freeadicals’ ability to denaturalize proteins and their low molecular weight,hey easily diffuse through enamel matrix and reach dentin (20). Wheneactive oxygen species radicals penetrate dentinal tubules, they have tovercome different defense mechanisms, including the odontoblastrocess, dentinal fluids, and collagen fibers, that act as a biologicalarrier by interacting with these molecules because of high reactivity ofadicals (34, 35). Moreover, positive intrapulpal pressure and gel osmoticressure could oppose to the diffusion of radicals toward the pulp (32).
In case the free radicals reach the pulp tissue, pulp cells have thebility to synthesize enzymes, like catalase, glutathione peroxidase, anduperoxide dismutase, which are part of the homeostatic system of theody and serve as a defense mechanism against peroxides and freeadicals. Additionally, synthesis of hemoxigenase-1 (HO-1) is increasedfter cell exposure to oxidative stress. HO-1 is a heat shock protein thatapidly responds in cases of aggressions that threaten cell homeostasis.dontoblasts have shown a strong positive staining for HO-1, which inart could be responsible for the apparent safety of tooth bleaching,ecause odontoblasts are the first cells that become in contact with freeadicals inside the dentinal tubule (7).
However, when the amount of highly reactive radicals (HO� and�) that penetrate tooth mineralized tissues overcomes these pulpo-entin complex initial defense mechanisms, irritation of nerve sensory
ibers occurs with the consequent neuropeptide release, triggering aore aggressive defense mechanism known as inflammation (1, 5, 10).
his could explain why light-/heat-activated tooth-bleaching systemsenerate an increased SP expression in the present study, whereas theggression of 38% H2O2 alone could be more tolerable or controlled byulp defense mechanisms.
Another possible explanation to the significantly higher SP levels inhe 25% H2O2 activated with UV light compared with the other groups ishe fact that H2O2 interacts intensely with lights of a wavelength smallerhan 300 nm (UV light range), generating a greater ionic dissociationnd an increased free radicals release (12).
The increased levels of SP found in the present study may belinically relevant because they could develop a neurogenic inflamma-ory reaction in pulp tissue, which may not be evidenced with severe orpontaneous pain because of the degenerative processes that free rad-cals produce over cells, nerve fibers, and blood vessels. Therefore, itan be concluded that light- and/or heat-activated tooth-bleaching sys-ems generate a significant increase in pulpal SP levels. These datahould make clinicians aware that performing this type of tooth-bleach-
ng procedure, even under the best conditions, represents an injury toJOE — Volume 34, Number 12, December 2008
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he pulpodentin complex, which could generate neurogenic and vascu-ar reactions, including the release of SP.
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