Pulp temperature is more related to radiant exposure values than to irradiance.
Higher rate of pulp temperature rise increased precursors of inflammatory response.
No distinct histological changes in pulp were noted when temperature increased 5.5 °C.
To evaluate the influence of light emitted from two Polywave®, LED light-curing units (LCU) on in vivo pulp temperature (PT) rise and signs of acute inflammatory response in pulps of human premolar having deep Class V preparations.
Sixty intact, first premolars from 15 volunteers requiring extraction received infiltrative anesthesia. A sterile thermocouple probe was inserted within the pulp tissue through a minute occlusal pulp exposure in only 45 teeth (n = 9) to continuously monitor PT (°C). A deep buccal Class V preparation was created, and the surface was exposed to light from a commercial Polywave LCU (Bluephase 20i (20i), Ivoclar Vivadent) or from an experimental LCU (Exp) using the exposure modes (EM): 1s/Exp and 2s/Exp, 10s/20i, 20s/20i, and 60s/20i. Peak PT and PT rise values above baseline (ΔT) data were evaluated using a one-way ANOVA followed by Tukey’s post-hoc test ( α = 5%). Teeth used for histological and immunohistochemical analyses (n = 3) were extracted approximately 2 h after exposure to the LCU.
No significant difference in peak PT and ΔT values was noted between 2s/Exp and 20s/20i groups, which both exhibited higher values than 1s/Exp and 10s/20i groups ( p < 0.001). Dilated and congested blood vessels were seen after exposure to 1s/Exp, 2s/Exp, or 60s/20i EMs. The expression of IL-1β and TNF-α tended to be more intense when higher irradiance was delivered.
Although higher irradiance delivered over a short exposure caused lower PT rise than 5.5 °C, such EMs should be used with caution, as they have more potential to harm the pulp tissue.
The use of light-curing units (LCU) is a daily routine in dental offices. Based on the market volume of light-cured composite resins sold annually, it is estimated that approximately 261 million composite resin restorations are placed every year [ ]. Among other reasons, these materials are widely used due to ease of application and availability, reduced chair time, and affordability [ ]. As a consequence, LCUs have become valuable tools in the clinicians’ daily routine.
Recently, manufacturers developed powerful light-emitting diode (LED) LCUs capable of providing radiant emittance values of over 2000 mW/cm², in order to reduce clinician chair time. It is commonly thought that such a high irradiance could be delivered for a shorter time interval and supposedly obtain a similar polymerization result. Heat generated at the light target is related to increased radiant emittance values, and these powerful devices generate considerable amounts of heat [ ]. This effect leads to higher temperature increase within the composite resin layer during resin polymerization [ , ] as well as within the pulp chamber, as previously shown in in vitro and in vivo studies [ ]. Although the temperature increase within the pulp chamber during light activation is attributed to both the exothermic reaction of polymerizing material and to the radiant energy absorbed during light exposure [ , ], most of the heat within the pulp chamber is attributed to exposure from the emitted light [ , ].
It is well known that heat can damage the pulp. According to a study commonly cited for correlating pulp temperature and subsequent pathological conditions of the human pulp (Zach and Cohen [ ]), if the pulp temperature (PT) of rhesu s monkeys increased to 5.5 °C, a subsequent rate of 15% of necrotic pulp tissues were observed. Based on these findings, and the lack of similar human-based trials, many authors consider a PT rise of 5.5 °C as the potential damaging threshold for human pulp tissue. However, other studies have shown controversial findings. For instance, some authors observed that a PT increase to 39–42 °C only caused a perceptible increase in the rate of pulp blood flow in rat incisors [ ]. Stasis and thromboses that led to circulation arrest were observed only when PT increased to 46–50 °C. Decades later, Baldissara et al. [ ] observed that a PT increase of 11.2 °C arising from a thermal impulse lasting between 1 min 40 s to 3 min was not capable of causing irreversible inflammatory changes in human pulp. The authors attribute differences in histological findings among studies to the higher rate of PT rise in the methodology described by Zach and Cohen [ ] compared to that described in the Baldissara et al. [ ] study. Therefore, those authors believed that the harmful influence of heat on pulp tissue was not only related to PT increase but also to how rapidly the heat passed from the tooth surface into the pulp, as well as the duration of the heat stimuli.
More recently, in vivo studies were performed on intact human premolars or on premolars having Class V preparations, and real-time PT increase during exposure to curing light emitted from a Polywave® LED LCU was measured at varying radiant exposure values and output modes [ , ]. In both studies, a positive, linear relationship between applied radiant exposure values and PT rise values was observed. In addition, tooth exposure to light having an irradiance of approximately 1230 mW/cm 2 for 60 s resulted in a PT rise close to or similar to that considered harmful for the pulp (using the Zach and Cohen model) [ ]. Furthermore, teeth exposed to light with higher radiant emittance value showed a more rapid PT rise than when the teeth were exposed to light having lower radiant emittance values [ , ]. Such findings deserve some concern, because manufacturers are constantly launching LCU devices having exposure mode (EM) options generating delivery at considerably high radiant emittance values for only a very few seconds. Therefore, PT rise caused by these EMs may be harmful to the pulp, not only because of the increase in PT but also because of the possible high rate of PT rise resulting from use of such EMs. However, to date, no information is available concerning the consequences of these EMs on PT increase and the first signs of acute pulpal inflammatory response in human premolars. In this regard, the in vivo analysis of PT rise during exposure to a curing light along with ex vivo histological and immunohistochemical analyses of extracted teeth to evaluate precursors of inflammatory response and histological signs of possible acute pulpal inflammatory changes are valuable tools to evaluate the impact of short exposure at high radiant emittance values on PT rise values currently considered harmful on the pulp tissue.
Thus, the purpose of this study was to evaluate the in vivo PT rise, histological changes, and the presence of acute inflammatory precursors arising after exposure of upper, human premolars having deep, buccal Class V preparations, to light with high radiant emittance values emitted by two Polywave® LED LCUs. The research hypotheses were that (1) PT increase would not be significantly different between use of Polywave LEDs delivering the same amount of radiant energy (one light doing so using low irradiance for a longer duration, the other light delivering a higher output for a shorter time); (2) there would be no significant difference in either histological findings or in levels of precursors of acute inflammatory response between use of Polywave LEDs delivering the same amount of radiant energy (same condition as for hypothesis one); and (3) PT levels similar to those considered harmful for the pulp (the Zach and Cohen standard values) leads to greater release of precursors of inflammatory response as well as acute inflammatory changes than in specimens where lower PT values are measured.
Material and methods
Overall study plan
Fig. 1 presents a graphical depiction of the overall research plan.
LCU output characterization
Two Polywave LED LCUs were used: one commercially available (Bluephase® 20i, Ivoclar Vivadent, Schaan, Liechtenstein) (20i) and one experimental LCU (Exp), which was exclusively built by Ivoclar Vivadent to provide extremely high radiant emittance values over very short exposure times. The spectral power of both LCUs was recorded five times each, using a laboratory grade spectroradiometer (USB 2000+, Ocean Optics, Dunedin, FL, USA) and a 6-in integrating sphere (Labsphere, North Sutton, NH, USA), previously calibrated using a NIST-traceable light source. Each LCU tip end was positioned at the entrance of the integrating sphere, such that all light emitted from the unit was captured. The EMs evaluated for Bluephase 20i and Exp LCUs were the High and Turbo modes, respectively. Wavelength-based, spectral power emission during each EM was determined using software (SpectraSuite v2.0.146, Ocean Optics) between 350 to 550 nm, which also provided a total of emitted power within that wavelength range. The optical emitting area of the distal end of the light guide was calculated, and this value was divided into the integrated spectral power value to derive the total radiant emittance from the curing light for each EM (mW/cm 2 ).
It is important to note that the radiant exposure generated using the 10s exposure from the Bluephase 20i unit and that from 1s of the Experimental LED unit were almost equivalent, despite the radiant emittance of the experimental light being 8 times greater. The same comparison is drawn to the Bluephase 20i used for 20s and the Experimental light used for only 2s. Exact equivalence of radiant exposure values could not be obtained, but close values were found when using the available, set exposure durations of the various LCUs. These relationships were those used to evaluate the first and second research hypotheses.
In vivo measurement of intrapulpal temperature
The method to measure real-time in vivo PT changes was based on previous studies [ , , ]. The study protocol was approved by the Ethics Committee at the State University of Ponta Grossa (protocol # 255,945). Fifteen volunteers requiring extraction of upper and lower first premolars for orthodontic reasons were recruited in the Orthodontic specialization programs in Ponta Grossa, State University of Ponta Grossa (UEPG) and at the Brazilian Association of Dentistry (ABO), Paraná section. Patient inclusion criteria included (1) treatment plans indicating premolar extractions for orthodontic reasons, (2) the presence of healthy, intact, non-carious, and non-restored, fully erupted treatment teeth, and (3) patients with well-controlled health conditions that allowed all procedures involved in the research to be performed with minimal risk. The subjects, ranging from 12 to 30 years (7 males and 8 females) went through an initial clinical exam at the UEPG clinics, after which the research methodology and study aims were explained. Following agreement to participate in the study, each subject signed a written informed consent, and the anamnesis and clinical record were filled in.
For the real-time PT analysis, 45 teeth were evaluated, while 15 premolars were used for the histological and immunohistochemical analyses. Therefore, 60 intact, first premolars teeth were tested in the current study. A single tooth at a time from a volunteer was treated by receiving infiltrative and intraligamental anesthesia, using approximately 1.8 mL of 2% Mepivacaine Hidrocloryde (36 mg) containing 1: 100,000 Epinephrine (18 μg) (Mepiadre, Nova DFL Industria e Comercio, Rio de Janeiro, RJ, Brazil). When testing only those teeth for which intrapulpal temperature data were recorded, following placement of a rubber dam, a small, occlusal preparation was made in the center of the tooth using a round diamond bur (#1013, KG Sorensen, Cotia, SP, Brazil) under constant irrigation until the preparation floor was near the roof of the buccal horn of the pulp chamber. A small, pencil-shaped diamond bur (#2134, KG Sorensen) was used to produce a minute pulp exposure at that location, which produced no pulpal bleeding.
A wireless, NIST-traceable, temperature acquisition system (Temperature Data Acquisition — Thermes WFI, Physitemp Instruments Inc., Clifton, NJ, USA) was used to measure PT. Two, 1-cm long tip, calibrated temperature probes (Model MT-D, Type T, time constant: 0.025 s, Physitemp Instruments Inc.) were connected to that system and both tip ends were immersed in a room temperature, 0.9% sterile saline solution, while tooth preparation was performed. After pulp exposure was obtained, one probe was removed from the water and immediately inserted into the pulp chamber through the occlusal access opening and was positioned to remain stable, while PT was measured. A small groove was created on a buccal cusp incline, close to the cusp tip, to allow the probe to rest securely, ensuring that the 1-cm long probe tip penetrated approximately 4 mm into the pulp chamber: in a similar position for all teeth. The other probe was kept in the saline solution at room temperature (approximately 22.0 °C), as a reference and to ensure consistent operation of the temperature measurement system. The room temperature was stabilized using air conditioning set to approximately 22 °C. The tooth preparation was then filled with provisional restorative material (Cavitec, Caithec Ltda, SC, Brazil), to minimize heat loss from the tooth through the occlusal preparation walls and pulp access. After probe stability was confirmed, real-time temperature data were continuously acquired every 0.2 s, until a stable PT was reached: between 15 min and 20 min.
Class V preparation
A controlled-size, Class V tooth preparation was made on the buccal surface, within the gingival 1/3 of the tooth, using a diamond bur (#2131, KG Sorensen, Cotia, São Paulo, Brazil) in a high speed hand piece, under controlled amounts of air–water spray (33 mL/min), as previously described [ ]. Water flow rate was measured by collecting fluid from the turbine in a graduated cylinder over measured time intervals. The preparation size was determined based on physical dimensions of the specific bur shape: 2.5 mm in diameter and 2.5-mm deep. The preparations left approximately 1-mm thickness of dentin remaining between the preparation axial wall and the pulp chamber. Following tooth preparation, another time interval was provided for the intrapulpal temperature to reestablish to a stable, physiologic baseline value.
Curing light exposure scenarios
Each LCU tip was placed against the buccal tooth surface, directly centered over the Class V preparation. Attention was made to establishing a similar relationship between the body of the LCU and light guide position, as well as the orientation of the tip end to the tooth. In this manner, any variance in beam homogeneity (both in terms of wavelength or local irradiance) would be controlled and made consistent. The tooth was randomly, sequentially exposed to the radiant output from the Bluephase20i unit using the following EMs (EMs): 10-s (10s/20i), 20-s (20s/20i), and 60-s (60s/20i) in the high intensity mode, allowing a 7-min span between each exposure, enabling the baseline PT to be reestablished before the next exposure (n = 9). When teeth were exposed to light emitted from the Exp LCU, the following EMs were used: 1-s (1s/Exp) and 2-s (2s/Exp) in the Turbo mode. The precise time into the data acquisition profile when each light mode was applied was recorded using a digital counter, so that an overlay and subsequent correlation of light activation and temperature rise could later be made.
At the end of temperature analysis, the probe was carefully removed from the tooth, which was extracted, following the patient’s orthodontic treatment plan. After extraction, the probe was reinserted into the pulp chamber of the extracted tooth, and X-ray images were obtained from the proximal side, with the probe in position as it was intraorally. This process confirmed that the probe was properly inserted into the pulp chamber during temperature measurement, and also provided an indication of the remaining dentin thickness of the Class V preparation.
Time constant analysis of each thermocouple was determined, as previously described by Runnacles et al. [ ]. In summary, the thermocouple was immersed in hot (≈60 °C) and room temperature water (≈25 °C), developing a temperature vs time plot, and the average time corresponding to 63.2% of the total temperature increase (time constant; τ) was determined. For the temperature data acquisition system, the τ obtained was 1.46 s. The slope of the regression line between two temperature extremes was used to determine the time required for the system to indicate a 1° Centigrade temperature change: only 0.07 s.
Exposure to light emitted from the LCUs in teeth for histological and immunohistochemical analysis
For the histological and immunohistochemical analyses, the same procedures and exposure modes were performed in teeth that were not previously used for the PT analysis. However, for these conditions, no rubber dam was placed nor was any occlusal preparation made or pulp exposure attained. In addition, three teeth having Class V preparations, but not having any exposure to light, were evaluated as the control group (n = 3). Immediately following tooth preparation in the control group, or after any exposure to curing light was performed, the tooth was restored using a self-curing glass ionomer (Vidrion R, S.S. White, Rio de Janeiro, RJ, Brazil). After approximately 2 h from time of restoration placement, the teeth were extracted in accordance with the patient’s orthodontic treatment plan.
Analysis of general histological features and precursors of pulp inflammatory response
The presence of specific inflammatory precursors were evaluated: IL-1β and TNF-α. The former pro-inflammatory cytokine is an important mediator that is crucial for host-defense responses to infection and injury, and is involved in a variety of cellular activities, including cell proliferation, differentiation, and apoptosis [ ]. The latter cytokine is involved in systemic inflammation and is a member of a group of such compounds that specifically stimulate the acute phase of the inflammatory response [ , ].
All extracted teeth were immediately fixed in 10% buffered formalin for 24 h. Later, the teeth were decalcified using a 4.13% EDTA solution at a pH of 7.0, and then embedded in paraffin. Semi-serial, longitudinal, 6-μm-thick sections were obtained and stained with hematoxylin and eosin (H&E) for routine histological examination under optical microscopy. Three-μm-thick sections were used for immunohistochemical staining to determine the expression of the previously mentioned inflammatory mediators. Tissue sections for immunohistochemical analyses were placed on glass slides previously treated with 3-Aminopropyl-triethoxi-silane. The antigen recovery process was accomplished by moist heat radiation in EDTA solution at pH of 9.0 for IL-1β and in 10 mM Citrate buffer (pH 6) for TNF-α. Afterwards, the slices were washed with 20 V Oxygen Peroxide to block endogenous peroxidase. The treated slices were subjected to 16-h of primary antibody incubation (overnight) at 4 °C and were then washed 3 times with PBS. The reaction was detected using a commercially available LSAB Staining System kit (Santa Cruz Biotech Inc., Texas, USA). The resulting slices were stained with Carazzi hematoxylin, subjected to baths in xylol, and mounted in Canada Balsam. To validate the methodology, some slices were subjected to all steps of immunohistochemical analysis, excepting primary antibody incubation. The positive control reactions were tested on tissues from a known periapical granuloma. Immunostaining for all specimens was evaluated along the entire pulp chamber extension using a conventional optical binocular microscope (CH2, Olympus, Shinjuku, Tokyo, Japan). Images from the sections were captured using a digital camera coupled with 400× magnification and analyzed in an image analysis program (ImageJ, 1.46r, National Institute of Health, USA).
Histological and immunohistochemical analyses were based on visual aspects observed in the pulp tissue after exposure to light emitted from the LCUs compared to visual patterns observed in the control group (no exposure to curing light). A single, trained pathologist was responsible for making and recording all observations. The histological analysis was performed to detect the presence of inflammatory signs and changes in pulp morphology regarding the odontoblastic layer and blood vessels. In the immunohistochemical analysis, the presence of immunostaining (brown-color) was evaluated over the entire pulp tissue area as an indicator of the expression of inflammatory mediators IL-1β and TNF-α.
For the parameters of peak PT and ΔT, the homogeneity test of variances and Shapiro Wilk normality tests were performed, and were passed. Thus, peak PT and ΔT data were evaluated using parametric statistics: a one-way ANOVA followed by Tukey’s post-hoc test at a pre-set alpha of 0.05. Post-hoc power analysis was also performed peak PT and ΔT values. The 95% confidence intervals of mean slopes of PT rise were determined and compared (Microsoft Excel 2007, Microsoft Corp., Redmond, WA, USA). The mean slopes were not significantly different when an overlap between confidence intervals was noted. All statistical analyses were performed using commercial statistical software (Statistics 19, SPSS Inc., IBM Company, Armonk, NY, USA).
For the number of evaluated teeth (n = 9), the in vivo study was adequately powered for both peak PT values and for ΔT values (over 99.0%; pre-set α = 0.05). Radiant emittance, radiant exposure, peak PT, and ΔT values are presented in Table 1 . Teeth exposed to 60s/20i EM showed the highest peak PT and ΔT values ( p < 0.001). The importance of comparisons to be made are that no significant differences in peak PT and ΔT values were noted between 2s/Exp and 20s/20i groups, which in turn exhibited significantly higher peak PT and ΔT values than 1s/Exp and 10s/Exp groups (p < 0.001), such were not significantly different between themselves. These specific scenarios were paired for comparison, because they represented near similar radiant exposures (10s/20i and 1s EXP (12.3 and 10.0 J/cm 2 , respectively), 20s/20i and 2s EXP (24.6 and 20.0 J/cm 2 , respectively)).