Assesment of radiotherapy effects on the blood flow in gingiva and dental pulp - a laser Doppler flowmetry study

Abstract Objective This study aims to determine and compare the dental pulp and gingival blood flow in patients referred for oropharyngeal radiotherapy (RT) at three different time points: before the start, immediately after, and six months following the completion of RT. The aim is also to evaluate the dependence of the pulp and gingival blood flow on the radiation dose. Methodology A prospective study included 10 patients referred for intensity-modulated RT (IMRT) in the oropharyngeal region, with at least one intact tooth surrounded by a healthy gingiva. The dose received by each selected tooth and adjacent gingiva was determined according to the map of treatment planning and computer systems. The blood flow measurements were performed using the laser Doppler flowmetry (LDF) method. Results Comparing vascular flows at three different time points, the median blood flow in the dental pulp showed no statistically significant difference (p=0.325), contrary to gingiva (p=0.011). Immediately after RT completion, the gingival flow significantly increased compared to its starting point (p=0.012). The pulp flow correlated negatively with the radiation dose, whereas a strong correlation was noted 6 months following the RT completion. Conclusions RT caused a significant acute gingival blood flow increase, followed by a long-term (over six months) tendency to return to the starting levels. The dental pulp blood flow is differently affected by higher radiation doses (over 50Gy) in comparison to lower doses (below 50Gy). During RT planning, considering the possibility of protecting the teeth localized near the Gross Tumor Volume as a sensitive organ is recommended.


Introduction
Currently, radiotherapy (RT) is an integral part of the treatment of various neoplastic processes.
Applied in the treatment of oral and oropharyngeal malignancies, RT can be responsible for many transient or permanent side effects in the oral cavity, such as oral mucositis, radiation caries, osteoradionecrosis, or alteration of salivary gland function. 1,2 By introducing 3D conformal planning and intensity-modulated radiotherapy (IMRT), surrounding healthy organs became more protected from its harmful effects. 2 Studies confirm greater sparing of the salivary glands and a lower rate of xerostomia when using modern RT modalities. [2][3][4] However, teeth with supporting tissues, located within the irradiated volume, are exposed to harmful effects to a greater or lesser extent, depending on the individual dose they received, which can differ from the tumor dose. RT also causes inflammatory changes in the oral mucosa and gingiva, which can result in necrosis, atrophy, or unusual gingival enlargement. 5 Atrophy of the gingiva leads to recession and painful sensitivity in the exposed tooth root, whereas gingival enlargement causes food impaction and periodontitis. Based on the fact that ionizing radiation causes physical and chemical tissue damages, some authors suggested that applied RT causes dose-dependent alterations of the vascular flow in dental pulps, with possible but not necessarily permanent consequences: fibrosis and atrophy. 6-8 Shenoy, et al. 8 (2007) have reported that the resulting changes in pulp tissue can give a false negative response to standard pulp sensitivity and vitality tests and indicate inadequate treatment: endodontic therapy or even tooth extraction, which can trigger a serious complication: osteoradionecrosis of the jaw. 8,9 So far, no correlation between the intensity of the acute effects and the severity of late RT sequelae has been proven, which hinders radiation damage The study was designed as a prospective study.
Data from the literature 10 were used to estimate the sample size for the difference between the means of the blood flow in gingiva and dental pulp before and after radiation therapy. The sample size consisted of 10 subjects to test the difference between the means for a significance level of 0.05 and a statistical strength of 80% in the program G*Power 3.1.9.4 (Germany).
Inclusion criteria 1) Patients with a diagnosis of oral/oropharyngeal cancer referred to RT who gave written "informed consent"" to participate in the study 2) Presence of an intact tooth with a healthy adjacent gingiva (without signs of inflammation) and with no history of previous painful sensations were compared between the groups that received up to 50 Gy and those that received more than 50 Gy, using the Mann-Whitney test. Spearman's coefficient was used to show the correlation of the radiation dose do the dental pulp and gingival flow. P values < 0.05 were set as significant.

Results
Due to the two dropouts during the study, eight respondents were included in the analysis (three men and five women), aged 32-68 years, with the average age of 50.75 years (post-hoc achieved power analyzed in G*power program version 3.1.9.2. was 96.29%). All respondents had tumors of the oral cavity, including several different but relatively close localizations: mucosa of the alveolar part of the mandible-1, mucosa of the alveolar part of the maxilla-1, buccal mucosa-1, base of the oral cavity-1, tongue-3, and tongue with the base of the oral cavity-1.
Of a total of eight tested teeth, three were central maxillary incisors, four were lower canines, and one was a lower premolar. Although the total radiation doses in all patients were 60-66 Gy, the individual doses received by the teeth and adjacent gingiva included in the study differed significantly and varied from 20 to 60 Gy. The dose for the tooth and associated gingiva corresponded to the total received dose (60Gy) in only two of eight patients. Four of a total of eight teeth received a dose greater than 50 Gy, whereas the remaining four received a dose of less than 50 Gy.
A descriptive analysis of gingival blood flows showed a significant increase in the median value at FT1 compared to FT0, followed by a decrease at the FT2, although no complete return to the starting point was found (Figure 1). Comparing the gingival flow values obtained at different measurement points, a significant difference (p=0.011) was observed.
Additionally, the Wilcoxon test was applied, which showed that the obtained significance comes from the difference in flow values FT0 and FT1 (p=0.012).
The dental pulp blood flow showed a slight increase of the median value starting from the initial FT0, over   (Figure 4). As opposed to this, regarding the doses of up to 50 Gy, a slight increase in these values prevailed, although without a statistical significance.
The radiation dose and gingival flow showed no statistically significant associations or a strong correlation. In contrast, a negative correlation between the pulp flow and the radiation dose at times FT1 and FT2 was observed, indicating that when the dose is increased, the dental pulp flow decreases (Table 1).
Although no statistically significant association was found, a strong correlation was observed six months after the completion of RT (FT2).   15 The increase in the oral hygiene index could be explained by the associated hyposalivation, pain, and trismus, but also by the lack of psychological and emotional motivation of oncology patients. 15 Gingival recession was more pronounced in the mandible than in the maxilla, which can be related to the lesser degree of vascularization.
In addition, an unusual enlargement of the gingiva several months after the end of RT was mentioned in the literature, whereas the multiplication of gramnegative bacilli is cited as a possible mechanism for its occurrence. 5 A change in the oral microflora and an increase in the level of endotoxin released by gram-negative bacilli could play an important role in the development of inflammatory changes in the gingiva and lead to an altered tissue response and consequent gingival enlargement. Poor oral hygiene undoubtedly supports and accelerates the process of periodontium alteration; therefore, dentists must educate their patients undergoing RT. Although a descriptive analysis of pulp flows in our study showed a slight increase in the median value after completed RT compared to the initial value, no statistically significant difference was observed. Kataoka, et al. 16 (2011) showed different responses of dental pulp circulation to RT using the pulse oximetry method. The authors observed a significant reduction in the pulp oxygenation level from the initial 93% before treatment to 77% at the end of the RT treatment (60-70Gy), including a repeated increase in %SpO2 values with a tendency to approach initial values (85%) 4-5 months after treatment. These differences could be explained by the fact that the study by Kataoka, et al. 16 (2011) was based on a different methodology (pulse oximetry against blood flow in our study), differences in the age structure of the examined patients, as well as probable differences in the received dose at the level of the examined teeth.
The authors did not evaluate the doses of radiation for the tooth per se. Furthermore, the average age of the patients was 47.2 years (the oldest patient was 55 years old), whereas in our study the average age was 50.75 years (the oldest patient was 68 years old).
Our results showed a decrease in the median pulp flow FT2 compared to the initial value of FT0 with doses > 50 Gy; however, an increase in these values prevailed with lower doses, although no statistical significance was found. Correlation results confirmed a negative association between the pulp flow and radiation dose, indicating that increasing the dose decreases the pulp flow, as a strong correlation noted six months after the completion of RT was found. The fine vascular network of the dental pulp, enclosed within the surrounding dentin and without collateral circulation, creates a low compliance circulatory system that is probably more sensitive to high doses of radiation compared to that in the gingiva. Although the studies of Kataoka, et al. 7, 16 (2016Kataoka, et al. 7, 16 ( , 2011  As limitation of our study we cite the sample size. Although a difference in the flow changes was observed after the end of therapy between the teeth that received <50 Gy and those that received >50 Gy, statistical significance was not reached. Knowing the fact that the pulp flow showed a strong negative correlation with the radiation dose six months after RT completion, it would be justified to conduct a comparative study of the pulp flow changes after completed RT between the teeth that received <50 Gy and those that received >50 Gy, with a larger sample. The strict exclusion criteria should not be ignored, as well as the fact that this is the first study that considered the variability of radiation doses for the tooth and adjacent gingiva, which is the reality of current RT modalities. The most demanding criterion, which a small number of patients aged 30-70 satisfy, is the presence of intact teeth and healthy gingiva.
Consequently, our sample consisted of teeth from different morphotypes (central maxillary incisors, lower canines, and lower premolar) that, according to Norer, et al. 17 (1999), could potentially affect PBF levels. We believe that this has no significant impact on our results considering the fact that we observed and compared blood flow levels of the same tooth at three-time intervals (before, after, and six months following radiation therapy). The studies by Kataoka, et al. 7,16 (2016Kataoka, et al. 7,16 ( , 2011 also considered teeth with smaller restorations, but we believe that any preparation and production of secondary dentin could affect the vascular flow.

Conclusion
Currently, highly conformal dose distributions and dose gradients in pathological and normal tissues, delivered by IMRT, have made the appearance, severity, and possible sequelae specific to each patient, requiring a strictly individual approach.
In gingivae, RT causes acute blood flow increase, followed by a long-term (over six months) tendency to return to the starting levels, whereas the dental pulp blood flow is differently affected by higher radiation doses (over 50 Gy) in comparison to lower doses (below 50 Gy). When planning RT, we recommend considering the possibility of protecting the teeth localized along the GTV as a sensitive organ to avoid permanent consequences, especially if the given teeth are perceived as potential carriers of future prosthetic restorations. Before starting RT, we advise a consultation with a dentist and, in indicated cases, pre-irradiation dental treatment.