Mechanical loading on the functional activity of carbonic anhydrase II in the periodontium

Carbonic anhydrase II (CA II) is involved with the acid-base homeostasis of tissue. This study aims to evaluate the effect of traumatic dental occlusion (TDO) by means of CA II expression in osteoclasts and osteocytes (near the lamina dura and in the centre of alveolar bone septum), in the periodontal ligament (PDL) and in lining cells (periosteum). For this study, 50 male Wistar rats aged seven weeks were divided into 2 groups: Loaded and Unloaded group. The study periods were 2, 5, 7, 14 and 21 days. The Mann-Whitney U test for quantitative, and the Chi-square test for semi-quantitative analyses were used for group comparison, along with Bonferroni’s post-hoc test. Statistically significant differences between the groups were observed in the number of osteoclasts in the lamina dura (days 5, 7 and 21); the alveolar bone septum (days 2 and 7); osteocytes near the lamina dura (days 2, 5, 7 and 14); and in the centre of the alveolar bone septum (days 2, 5, 7 and 14). There were also differences between-group in CA II expression in the lining cells on days 7 and 14. TDO increases CA II expression in osteoclasts, osteocytes, the PDL and lining cells of the periosteum. Clinical Relevance: Research, Society and Development, v. 9, n. 9, e686997719, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i9.7719 3 Traumatic dental occlusion stimulates higher cells activity of the alveolar bone at short (lamina dura) and long (centre of alveolar bone and periosteum) distances.


Introduction
Mechanical stimulus is necessary for the maintenance of periodontal tissue homeostasis, whereas the absence or excess of occlusal load results in a disharmonic functioning of periodontal tissues (Glickman, 1971;Brandini, et al., 2018).
Primary occlusal trauma has been defined as an injury to the attachment apparatus as result of excessive occlusal force applied to a tooth or teeth with normal and healthy support tissues (Davies, et al., 2001). It can affect tooth mobility (Harrel & Nunn, 2001), fremitus, persistent discomfort or pain on eating (Wan, et al., 2012) and aseptic pulp necrosis.
In the first 24 hours of traumatic dental occlusion (TDO), osteogenesis in the rats, alveolar bone was inhibited, and osteoclastogenesis was not significant (Wan, et al., 2012).
An increase in the pressure of the interstitial fluid of the PDL was observed after 48 hours (Palcanis, 1973;Brandini, et al., 2018). Until the 5 th day the PDL space width decreased, and there was an increase in the number and a disorientation (Glickman, 1971;Palcanis, 1973;Brandini, et al., 2018) of fibroblasts, cell necrosis and venous thrombosis in the PDL (Palcanis, 1973), a decrease in collagenous fibres and elevated osteoclast activity (Glickman, 1971;Palcanis, 1973;Kaku, et al.,2005;Brandini, et al., 2018 ). There was also a significant increase in Ruffini endings and free nerve endings in the PDL. The PDL space returned to normality on day 7 (Kaku, et al.,2005), probably due to bone remodeling (Glickman, 1971;Brandini, et al 2016;Brandini, et al 2018).
CA II is involved with acid-base homeostasis. It provides the proton source for extracellular acidification by H+-ATPase and the HCO3-source for the HCO3-/Cl-exchanger (Teo, et al., 2012), and it catalyzes the reversible hydration of carbon dioxide into bicarbonate. It plays a critical role in the cells as a high neuronal activity in the periodontal Ruffini endings (Ochi, et al., 1998) and a decreased differentiation of ameloblasts (Wang, et al.,2010). Whereas some transporters are responsible for the bone resorption process, others are essential for the pH regulation in osteoclasts (Lehenkari, et al.,1998;Oksala et al., 2010).
CA II expression was found in the early stage of the osteoclast differentiation (Lehenkari, et al.,1998;Oksala et al., 2010). Bone resorption is stimulated by pH reduction in the osteoclast. Osteoclasts are multinucleated bone-reabsorbing cells that use multiple pH regulation mechanisms to create an acidic pH in the resorption lacuna (Riihonen, et al.,2007;Jansen, et al.,2009). Under conditions that favors bone resorption, osteoclasts reabsorb bone by attaching to the surface and then secreting protons into an extracellular compartment formed between osteoclasts and the bone surface. This secretion is necessary for bone mineral solubilization and the digestion of organic bone matrix by acid proteases (Jansen, et al.,2009).
The question now is to what extent traumatic dental occlusion can affect homeostasis of the periodontal tissues and maxillary bone.
This study aims to evaluate the effect of traumatic dental occlusion by means of CA II expression in osteoclasts and osteocytes the periodontal ligament and in lining cells of the periosteum. Research, Society and Development, v. 9, n. 9, e686997719, 2020 (CC BY 4.

Materials and Methods
First, approval was obtained from the Animal Care Committee of the Dentistry School of Araçatuba-UNESP (Process-2012-00980).
For this study, 50 male Wistar rats (Rattus Norvegicus, albinus) aged seven weeks were used. They were originating from the central bioterium of the Dentistry School of Araçatuba, the animals were transferred to the bioterium of the Department of Surgery and Integrated Clinic 5 days prior to the experiment. They were kept in cages with five animals each and given granulated food and water ad libitum. The environment was kept at a constant temperature of 22 º C (±2 ºC) and 50% (±10%) humidity, with light/dark cycles of 12/12 hours.
To assess the influence of mechanical loading on the alveolar bone of the right first upper molar following traumatic dental occlusion, the animals were divided into a Loaded (L) and Unloaded (U) group.
The periods of study were 2, 5, 7, 14 and 21 days for both groups. Group U group consisted of 25 same-aged ratsanimals with the same age as those in the experimental group, in order to enable comparison with normal conditions. Group L was comprised of 25 rats, submitted to mechanical loading on the occlusal surface of the teeth by placing a composite filling in the right inferior first molar.
In group L, the right first inferior molar was raised with a direct filling using 37% Research, Society and Development, v. 9, n. 9, e686997719, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i9.7719 For quantitative analysis, the images were processed using ImageJ. The number of osteoclasts was counted in the lamina dura around the distal root and in the centre of the alveolar bone septum in a selected area. The percentage of osteocytes was calculated in a limited area near the lamina dura and in the centre of the alveolar bone septum.
The semi-quantitative analysis of the entire PDL and lining cells of the periosteum used scores from 1 to 4, based on the color intensity of the CA II expression. The CA II expression in the lining cells of the periosteum was present on the vestibular side of the upper jaw in the correspondent region of the right first upper molar; and in the PDL of the right first upper molar's distal root.
The examiner was not informed to which group the images belonged, in order to avoid bias during the analysis.
The data were analysed with SPSS 20.0 (IBM, Armonk, NY, USA) at α=0.05. The Mann-Whitney U test was used for group comparison on the number of osteoclasts and osteocytes, and data were expressed as mean ± SD and percentage, respectively. The Chisquare test was used in combination with Bonferroni's post-hoc test for semi-quantitative analyses of the PDL and the lining cells, with data expressed in percentages of the score prevalence.

Results
In the group L on day 2, the CA II expression was significantly higher in the osteoclasts ( Figure 3) and osteocytes (Figures 1e, 1f, and 5) when compared with group U, located in the alveolar bone septum. Research, Society and Development, v. 9, n. 9, e686997719, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i9.7719 Figure 1. Transversal histological sections of the right upper first molar and surrounding tissue were stained with CA II. CA II positive osteoclasts (arrows) on day 5, the bone resorption and number of osteoclast (arrow) and osteocytes (arrows) can be seen in the group U (1a, 1c and 1e) and L (1b, 1d and 1f) (Magnification 200x). The presence of active osteoclasts (arrow) and the expression of CA II in the lamina dura can be compared between group U (1a) and L (1b) group. Detection of CA II positive osteocytes (arrows) on 2 day of the experiment in group L (1e) is more evident than in group U (1f). The lining cells show higher CA II expression (star) in group L (1h) than in group U on 14 day. (Magnification 400x).  (Figures 1a and 1b) indicated a higher Research, Society and Development, v. 9, n. 9, e686997719, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i9.7719 9 number of osteoclasts than on the day 2in the alveolar bone (Figures 1c and 1d), as well as in the lamina dura of group L (Figures 1a and 1b).  Research, Society and Development, v. 9, n. 9, e686997719, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i9.7719   After 7 days, the CA II expression became less intense, although the quantitative difference between groups L and U was still evident in the osteoclasts (Figures 2 and 3  On day 14 of the experiment the between-group difference in CA II expression had decreased, but could still been seen in the osteocytes (Figures 4 and 5) and lining cells (Figures 1g, 1h and Table 2).
The group difference had further reduced on day 21. The CA II expression showed a slight increase in osteoclasts ( Figure 3) and osteocytes ( Figure 5) in the centre of the alveolar bone septum. However, the number of osteoclasts in the lamina dura was higher in the group U ( Figure 2).

Discussion
Traumatic dental occlusion increases CA II expression in the PDL cells, osteoclasts, osteocytes and lining cells of the periosteum. The communication between these cells may be the network that preserves the homeostasis of the alveolar bone and periodontal connective tissue in the case of excessive mechanical loading.
Although the literature indicates that the PDL space returns to normal 7 days (6) (Islam, et al., 1990), and are even able to stimulate osteoclast precursors through RANKL expression (Tanaka, et al., 2000). In addition, they can act as bone surface cleaners through matrix metalloproteinase (MMP) activity prior to osteoclastic bone resorption (Riihonen, 2010). This might explain the relation between bone exostosis and occlusal forces (Yoshinaka, et al., 2014).
As a limitation of this study, can be considered the sample size, 5 animals per group in each period; and the semi-quantitative statistical analysis of some variables.
These observations expand the knowledge about the effects of traumatic dental occlusion. The intensity and frequency of traumatic dental occlusion affect the network between the cells, and may lead to temporary or permanent changes in the periodontium caused by the mechanical stimuli applied to cells. Long term studies are needed to identify all the alterations that can be caused by this type of trauma. CA II affects the resting intracellular pH, but the effect differs per cell type; meaning that this effector depends on cell action, not on CA II itself.

Conclusion
So, traumatic dental occlusion causes a significant increase in the CA II expression in osteoclast, osteocytes and in lining cells of the periosteum.