Microcirculation changes in gingival tissue after ultrasonic tooth preparation in beagle dogs

Abstract Ultrasonic wave technology is widely used during dental treatments. We previously demonstrated that this method protects the gingival tissue. However, the physiological change on the gingival microvasculature caused by this method remains unclear. Objective The aim of this study was to investigate the relationship between the morphological and physiological effects on gingival microcirculation when preparing teeth, using the conventional dental turbine or ultrasonic method. Methodology The lower premolar teeth of beagle dogs were prepared along the gingival margin by using a dental turbine or ultrasonic wave instrument. Gingival vasculature changes were investigated using scanning electron microscopy for corrosion resin casts. Gingival blood flow at the preparation site was determined simultaneously by laser Doppler flowmetry. These assessments were performed immediately (Day 0), at 7 days and 30 days after tooth preparation. Results At day 0, in the turbine group, blood vessels were destroyed and some resin leaked. Furthermore, gingival blood flow at the site was significantly increased. In contrast, the ultrasonic group demonstrated nearly normal vasculature and gingival blood flow similar to the non-prepared group for 30 days after preparation. No significant alterations occurred in gingival circulation 30 days after either preparation; however, the turbine group revealed obvious morphological changes. Conclusions Based on multiple approach analyses, this study demonstrated that ultrasonic waves are useful for microvascular protection in tooth preparation. Compared with a dental turbine, ultrasonic wave instruments caused minimal damage to gingival microcirculation. Tooth preparation using ultrasonic wave instruments could be valuable for protecting periodontal tissue.


Introduction
Ultrasonic wave technology is commonly used during dental treatments. For example, an ultrasonic scaler is used for periodontal and endodontic treatment, 1 whereas an ultrasonic knife is used during oral surgery. 2 Tooth preparation is typically conducted 0.5 mm under the gingival margin. When using rotating instruments (e.g., dental air turbine), a part of the gingiva is damaged, thereby causing bleeding.
Therefore, ultrasonic instrument is useful for tooth preparation since it maintains the margins in better condition. 3,4 Furthermore, this instrument provides sufficient dentin-cutting capacity without injuring the marginal periodontium. 5 We previously demonstrated that this method protects the microcirculatory system in the gingival tissue. 6 However, its influence on the physiology of the gingival microvasculature remains unclear.
The gingival microvascular network has been investigated using animals in previous research. 7 The formation of looping structures in gingival microcirculation could protect against bacterial invasion. 8 In addition, the morphology of the microvasculature of gingival tissue has been observed by using the India ink method. 7,9 The corrosion resin cast technique is widely available for observation of blood vessels in peripheral tissues, such as the gingival. 10-13 This technique can clearly obtain an image of the vasculature, including capillaries, by using a scanning electron microscope (SEM). The resin cast accurately reproduces changes in gingival circulation. 14 In addition, it has been demonstrated in human and animal studies that the gingiva blood flow can be changed by inflammation. 8 The laser Doppler flowmetry (LDF) is a non-invasive method that can be used to estimate the hemodynamics of microcirculation, and is an accurate and reliable method for assessing other microcirculation characteristics. This approach is widely utilized in pharmacological and/or physiological areas of research involving allergy testing, wound healing, and dermatosis. 15 While the skin is possibly the most studied organ, using LDF, it is also used to investigate internal organs such as the kidneys, liver, muscles, intestines, and brain. 16-21 Furthermore, previous reports have demonstrated that LDF is highly reproducible and enables blood flow to be measured in various environments. [16][17][18][19][20][21] Recently, the LDF has been applied to determine blood flow alterations with high reproducibility in the maxillofacial region, including the gingival mucosa, and gingival vascular function. [22][23][24][25] The aim of this study was to determine whether ultrasonic preparation led to alterations in blood flow.
We investigated the morphological and physiological effects of different tooth preparation methods -a conventional dental turbine or an ultrasonic instrument -on gingival microcirculation.

Methodology Animal subjects and tooth preparation
In this study, eight female beagle dogs (Oriental Kobo, Tokyo, Japan) had clinically healthy periodontal tissue; their teeth had undergone scaling 14 days before the surgery. The sample size for each group was six dogs -two dogs were operated in each day -for both two methods of tooth preparation. The control group (healthy gingiva) was composed by two dogs, which received no treatment. The teeth were prepared, as described in previous study. 6 Briefly, under general anaesthesia with an intravenous injection of pentobarbital sodium (25 mg/kg, SOMNOPENTYL; Kyoritsuseiyaku Co., Ltd., Tokyo, Japan), the lower premolar teeth were prepared along the gingival margin. On one mandible, the premolar teeth were prepared with a diamond bur (SF102R, Shofu Inc., Kyoto, Japan) and a dental turbine (MIJET-T Yoshida, Tokyo, Japan) with water spray ( Figure 1A); on the other mandible, the premolar teeth were prepared with ultrasonic waves (Suprasson P-max; Satelec, Merignac, France) using a diamond tip (FLT tip; Hakusui Trading Co., Osaka, Japan; Figure 1B). To ensure that the two methods of tooth preparation were comparable, they were performed by the same dentist. Physiological procedure Before conducting the morphological examination, gingival blood flow was determined at two points (mesial and centre) of eight lower premolar teeth at the same sites as tooth preparations using a dental turbine (four premolar teeth on one mandible) or ultrasonic waves (four premolar teeth on the other mandible).
Moreover, gingival blood flow was determined in the non-prepared control animals at the equivalent sites.
All gingival blood flows were estimated using an LDF

Statistical analyses
Statistical analyses were performed using GraphPad Prism (version 6.05.; GraphPad Software, La Jolla, CA, USA). Values are expressed as the mean±standard error of the mean. Data were analyzed using the twoway (methods of preparation × days after operation) variance analysis, followed by the Tukey's test. A P-value<0.05 was statistically significant.

Oral view
On Day 0 of tooth preparation, bleeding occurred along the margin (arrows) in the turbine group.
In the ultrasonic group, a vascular loop (arrows) was formed along the finishing line ( Figure 1D). On Day 7, blood vessels were extended in the turbine group

Morphological observation with scanning electron microscopy and hematoxylin and eosin staining
To confirm the state of the tooth surface, based on the preparation method, the cutting surface was observed using an SEM, haematoxylin, and eosin staining. In the dental turbine group, irregular and lateral stripes were observed (Figures 2A and 2B).  The dentition on the one mandible was prepared by a diamond bur and dental turbine with water spray. B: An ultrasonic device using a diamond tip. The dentition on the mandible was prepared by ultrasonic waves using a diamond tip with water. C: On Day 0, in the turbine group, blood vessels are extended with bleeding (arrows). D: On Day 0, in the ultrasonic group, a vascular loop (arrows) was continuously formed along the finishing line. E: On Day 7, in the turbine group, blood vessels are extended (arrows).

Thirty days after tooth preparation
In the dental turbine group, the blood vessels were creating U-turn loops in the marginal gingiva ( Figure   4E), these vessels were flat and thick (diameter 50-80 µm). In the ultrasonic group ( Figure 4F), most part of the vascular networks were arranged regularly. The marginal gingiva formed dense U-turn loops, which were similar to those in the control group (diameter 10-20 µm).

Physiological observations: gingival blood flow evaluated with laser doppler flowmetry
Gingival blood flow at the same site that the tooth preparation was significantly increased in the dental turbine group immediately (Day 0, P<0.05) and were recovered to same level observed in the control group after 30 days of tooth preparation ( Figure 5).   Otherwise, the ultrasonic wave group had no significant alteration in gingival blood flow in the preparation site at Day 0 nor 30 days after preparation ( Figure 5).
To record, the values of gingival blood flow were as follows: turbine group at 7 days, 36.32 mL/min/100 g; turbine group at 30 days, 36.45 mL/min/100 g; control group, 28.82 mL/min/100 g; ultrasonic group at 0 days, 27.75 mL/min/100 g.

Discussion
In this study, the gingival microcirculation changes that were induced by different tooth preparation methods were assessed by morphological and physiological alterations. In the chronological assessment of tissue damage after turbine or ultrasonic preparation, morphological alterations in the vasculature due to inflammation were apparent, although cases may occur without remarkable traits in gingival blood flow. Moreover, there may be no differences in blood flow among healthy, inflamed and in healing process gingiva. Our previous study recently proved that functional analyses using a single index may not be accurate and should be accompanied by appropriate morphological analyses. 22 In this study, we demonstrated, by using morphological and physiological approach, that ultrasonic wave instruments cause minimal damage to gingival microcirculation, compared with a dental turbine.
Injection methods are typically used to observe the vasculature. 6,26 The arterioles in the sulcular and junctional epithelium (JE) form a capillary network. 6 Because of the vascularization importance for tissues regeneration, it is essential to protect the marginal gingiva during tooth preparation. 6 Resin leakage, which indicates damaged blood vessels, 6  ( Figure 4D and Figure 5). These findings suggest that ultrasonic preparation does not disturb the gingival vasculatore, its circulatory systems nor induce the thermal damage that occurs after turbine preparation.
A limitation of this study is that no significant differences were observed between groups on days 7 and 30 after tooth preparation. This study demonstrates the safeness of ultrasonic preparation, and the findings are similar to those of a previous study. 6 Furthermore, the operability and clinical applicability of ultrasonic preparation were similar to those of dental turbine. Possible damage induced by ultrasonic preparation may have a minimal effect on the condition of periodontal tissues. It is well-known that the retention of a dental prosthesis on a dental abutment after tooth preparation is closely related to the periodontal status. Therefore, future studies are needed to investigate whether different methods affect the long-term retention of dental prostheses. Future studies are needed to clarify the effect of these factors on periodontal tissue.

Conclusions
By using multiple approach analyses, this study demonstrated that ultrasonic waves are useful for tooth preparations to minimize damage to the gingival tissue. Protecting the marginal gingiva during tooth preparation is essential since vascularization is important for tissue regeneration. Destruction of the JE causes periodontal disease, which suggests that tooth preparation using ultrasonic wave instruments is essential for protection of the periodontal tissue.