Comparison of in vivo and in vitro models to evaluate pulp temperature rise during exposure to a Polywave® LED light curing unit

Abstract Objectives: To measure and compare in vivo and in vitro pulp temperature (PT) increase (ΔTEMP) over baseline, physiologic temperature using the same intact upper premolars exposed to the same Polywave® LED curing light. Methodology: After local Ethics Committee approval (#255,945), local anesthesia, rubber dam isolation, small occlusal preparations/minute pulp exposure (n=15) were performed in teeth requiring extraction for orthodontic reasons. A sterile probe of a temperature measurement system (Temperature Data Acquisition, Physitemp) was placed within the pulp chamber and the buccal surface was sequentially exposed to a LED LCU (Bluephase 20i, Ivoclar Vivadent) using the following exposure modes: 10-s low or high, 5-s Turbo, and 60-s high. Afterwards, the teeth were extracted and K-type thermocouples were placed within the pulp chamber through the original access. The teeth were attached to an assembly simulating the in vivo environment, being similarly exposed while real-time temperature (°C) was recorded. ΔTEMP values and time for temperature to reach maximum (ΔTIME) were subjected to two-way ANOVA and Bonferroni's post-hoc tests (pre-set alpha 0.05). Results: Higher ΔTEMP was observed in vitro than in vivo. No significant difference in ΔTIME was observed between test conditions. A significant, positive relationship was observed between radiant exposure and ΔTEMP for both conditions (in vivo: r2=0.917; p<0.001; in vitro: r2=0.919; p<0.001). Conclusion: Although the in vitro model overestimated in vivo PT increase, in vitro PT rise was close to in vivo values for clinically relevant exposure modes.


Introduction
Maintenance of pulp safety is an essential challenge for clinicians in many restorative treatments, since heat generated from use of high and low speed handpieces, 1 restorative materials having exothermic setting reactions, 2 restoration finishing and polishing, 3 as well as from application of high power light emitting diode (LED) -based light curing units (LCUs) and laser sources to polymerize resin-based materials 4 may cause pulp temperature (PT) to rise to values considered harmful for the pulp. 5 For these reasons, in vitro temperature increase within pulp chamber of extracted teeth has been investigated. 4,6 Over the last decade, heat generated during tooth exposure to light emitted by LED LCUs has become an area of concern for clinicians and researchers. These concerns are based on availability of new, powerful light-curing devices that are capable of emitting light with radiant emittance values exceeding 2,000 mW/cm 2 . 7 Several studies evaluated the thermal stimulus caused by LED LCUs. Most of those studies relied on in vitro techniques using extracted teeth to evaluate temperature rise within the pulp chamber of extracted teeth while external heat sources were applied. 6,8,9 The most common methodology uses thermocouples inserted inside pulp chambers of extracted teeth to measure temperature changes in this location during exposure to various LCUs. 6,8,10,11 In an attempt to simulate the same physiological conditions observed in vivo, some authors developed specific devices in which the roots of extracted teeth were connected to a pump to provide a water fluid flow inside the pulp chamber so blood flow could be simulated, while the temperature inside the pulp chamber was initially stabilized at an average value close to the body core temperature (approximately 37°C) 6,8,12 or lower. 10,11 Conversely, other studies focused only on measuring temperature changes during exposure to LCUs, without simulating tooth physiological conditions. 9,13 Because of differences between these approaches, along with variance in LCU types, radiant emittances, and characteristics of teeth, 4,9,11,[13][14][15][16][17][18] a wide range in temperature value increases inside the pulp chamber, ranging from 1.5 to 23.2°C, is found in the literature. 4,9,11,[13][14][15][16][17][18] However, despite such differences in results and methodologies, many in vitro studies concluded that the use of some LED LCUs can cause an increase in temperature values within the pulp chamber higher than the threshold temperature increase considered harmful for the pulp (5.5°C). 5 Despite the important impact of these conclusions based on in vitro methods on the attention of researchers and manufacturers to this possible issue, it is reasonable to assume that in vitro conditions do not fully reproduce the complex physiological mechanism involved in the real in vivo condition. As a consequence, in vitro analysis is expected not to be capable of precisely reproducing in vivo PT when intact teeth are exposed to a LED light using varying exposure modes. However, due to the lack of in vivo studies that evaluated PT changes during heat stimulus when most in vitro studies were published in the past, and because of differences between tested teeth and tooth condition among studies, no contemporary data are available to confirm how well an in vitro model can reproduce temperature changes seen in the in vivo model, when under thermal stimuli such as the exposure to light emitted from a powerful LED LCU. Recently, an in vivo methodology was published that measured PT within the pulp tissue of human premolars. [19][20][21] In that approach, the temperature probe of a wireless temperature acquisition system, previously calibrated using National Institute of Standards and Technology (NIST-traceable) methods, is inserted within the human pulp tissue through an occlusal access, and real-time PT is monitored during thermal stimuli.
Thus, the purpose of this study was to evaluate how similar an in vitro model is able to reproduce temperature increase (ΔTEMP) values compared to the in vivo model, in anesthetized intact, unrestored, human upper premolars, in order to validate the in vitro methodology. The unique feature of this work was that the same premolar teeth tested for in vivo temperature rise were extracted for orthodontic treatment, and were subsequently tested in a clinically relevant in vitro system. In addition, the same LCU used in the in vivo analysis was also used for in vitro analysis. The tested alternative hypotheses were that   so that all light emitted from the unit was captured.
Wavelength-based, spectral and power emission during each EM were recorded using software (SpectraSuite v2.0.146, Ocean Optics) between 350 to 550 nm, which also provided the total emitted power value for that wavelength range. Radiant emittance values of each EM (mW/cm 2 ) were determined as the total measured

In vitro analysis of PT increase
The same premolars and LCU used in the in vivo study were tested in the in vitro, so any possible difference between outcomes would be exclusively attributed to the differences between the two models.
The extracted teeth were stored in 0.1% thymol (Symrise GmbH, Holzminden, Germany) until the moment they were fixtured to and tested in the in vitro model previously established. 10 In that approach, a test assembly simulated the in vivo environment:

In vivo and in vitro ΔTEMP and ΔTIME during curing light exposures
For the number of evaluated teeth (n=15), the in vivo study was adequately powered for EM and condition (in vitro and in vivo) factors (over 99,0%; α=0.05). Table 1  Despite the in vivo ΔTEMP average values being lower than 5.5°C, some teeth exhibited higher ΔTEMP values than that threshold temperature (Table 1)   values dropped immediately after the LED light shut off (Figures 3g and 3h). In teeth exposed to the 5-s/T EM in vitro, approximately half of the total PT increase occurred during exposure to the LED light, while the other half was noted after the light shut off (Figure 3f).

Discussion
To the extent of our knowledge, this is the first Curiously, despite such differences, no significant difference in ΔTIME was observed between both test conditions, even when in vitro ΔTEMP was 1.6 times higher than in vivo values. Such a finding infers that the in vitro rate of PT increase was higher than that Therefore, the first alternative hypothesis, stating that there are no significant differences in ΔTEMP values and ΔTIME measurements between in vitro and in vivo models, was partially rejected. Because the LED LCU, EMs and teeth were the same for both test conditions, such divergence between these results may be attributed to the dynamic regulatory mechanism of pulp tissue for heat distribution during temperature changes in this tissue used to dissipate heat transferred by external thermal stimuli throughout the dentine/ pulp complex. 6,25,26 In other words, when any external thermal stimuli generates more heat, fluid movement, either inwards or outwards from the pulp, will increase in an attempt to reduce the magnitude of PT rise. 6,25,26 For this reason, the actual in vivo pulp regulatory system has shown to be more effective in dissipating

Conclusions
Within the limitations imposed by the methodologies used, the following conclusions may be made: 1-The in vitro model detected higher PT increase than the in vivo model, when the same teeth were