Noengki Prameswari*, Christina Ajeng, Harum Azania, Clara Leona Orthodonti Department, Dentistry Faculty, Hang Tuah University
*Corresponding author: [email protected]
Abstract
Background: Maxillary transverse discrepancy usually requires expansion of the palate by a orthodontic tooth movements. Expansion appliance made the midpalatal suture that located between the maxillary bones in the palate is highly responsive to bone remodelling. Expansion appliance that can influence tooth movement and opening maxilla suture might affect to bone cells. Research about hyperbaric oxygen therapy during maxillary suture expansion has minimally investigated. Purpose: To analyzed balancing osteoclast/osteoblast in bone remodeling induced by giving HBOT 2,4 ATA given from day 8-14 to bone surface area during maxillary suture expansion. Material And Method: 18 healthy male guinea pigs with age between 2-3 months and weight range 200-300 grams were used in this research. With randomized post test only control group design divided into three groups: Normal control group K(O), negative control with expansion appliance K(-), hyperbaric oxygen therapy (T). After 14 days guinea pig decapitated. Midpalatal suture were cut horizontally and prepared into slides. Histological slide of osteoclast number, osteoblast number, and bone surface areas were seen and counted under light microscope with 400X magnification.
Statistically were analyzed by descriptive test, Kruskal Wallis and regression test. The level of significance was used in this study was settled at 0.05. Results: Descriptive test showed increasing mean of osteoclast cell number in K(0), K(-), and T was (0±0; 0.2±0.1; 1.7±0.1), mean of osteoblast cell number (14.9±0.2;
20.2±0.4,38±0.8); mean of osteoclast/osteoblast ratio (0.0±0.0; 0.01±0.0; 0.05±0.0) and bone surface area (0.7±0.07; 0.8±0.1; 1.1±0.1)induced by HBOT during maxillary suture expansion. Kruskal Wallis test showed that there is significance difference between each group. Regression test to examined the correlation between osteoclast number, osteoblast number, osteoclast/osteoblast ratio to the bone surface area.
Regression test showed that there is strong positive correlation between osteoclast number, osteoblast number, osteoclast/osteoblast ratio to the bone surface area with coefficient of multiple determination R=6.64 in HBOT during maxillary suture expansion. Discussion: Tight coupling of bone-forming osteoblasts and bone-resorbing osteoclasts occurs consecutively at a constant functional site. Imbalance osteoclast/ osteoblast ratio can disturb bone remodelling during maxillary suture expansion. HBOT have mechanisms to increase osteoblast, osteoclast, and bone surface area during maxillary suture expansion related to higher oxygen exposure can increase vascularization imply to bone cells. Conclusion: HBOT have role to increase osteoblast number, osteoclast number, osteoclast/osteoblast ratio, and bone surface area during maxillary suture expansion. There is intercausal relationship between increasing osteoclast number, osteoblast, osteoclast/osteoblast ratio inducing by 2,4 ATA HBOT to bone surface area during maxillary suture expansion.
Keywords: HBOT, maxillary suture expansion, osteoclast, osteoblast, bone surface area.
INTRODUCTION
One important treatment in orthodontic is correcting transverse maxillary deficiency. A number method to expand the maxillary, one of them is maxillary expansion. Orthodontic correction of maxillary constriction can be challenging as facial growth in the transverse dimension decreases during late childhood (Utreja, 2018). Clinical simptoms which maxillary expansion should be considered are crossbite, distal molar movement, maxillary retrusion, crowding (Gill, 2004). The purpose to correct the transversal discrepancy avoiding future extraction (Lagravere, 2005).
Midpalatal expansion, or rapid palatal expansion, has been used clinically with orthodontist for more than 40 years (Agarwal et al, 2010). There are many kinds of maxillary expansion appliances and various recommended to imply expansion rates, such as rapid maxillary expansion (RME), semi rapid maxillary expansion or slow maxillary expansion (SME) (Lagravere, 2005; Utreja, 2018).
Caprioglio study proved that mechanical stress during maxillary expansion resulting difference dimensional in the midpalatal along with radiographic examination, morphologic and histologic study. Mechanical separation is multiple step healing process involving bone cells indicated by new bone and connective tissue formation, followed by remodelling (Caprioglio, 2017). The underlying process that modulate bone formation and bone resorption during healing process of maxillary expansion are minimally discussed, but it has been reported that tensional force applied to the midpalatal suture of rats induces the replacement of cartilaginous tissues by bone (Agarwal et al, 2010).
When the maxilla is expanded, the mesenchymal cells placed on the inner side of the cartilaginous tissue proliferate and differentiate into osteoblasts (Agarwal et al, 2010). Remodelling process in maxilla expansion focuses on balance of bone cells.
Main point of application is the ratio of osteoblasts and osteoclasts in the bone multicellular unit (Heinneman, 2016).
Hyperbaric Oxygen Therapy (HBOT) is a method of medical treatment by inhaling pure 100%
oxygen continuously to the body with the air pressure with an ambient pressure higher than atmospheric pressure over specific time (Huda, 2010). HBOT increases the amount of oxygen that can be dissolved in a patient’s blood serum which states that the amount of ideal gas dissolved in solution is directly proportional to its partial pressure has been proven to
have beneficial effect for decompression illness and intended to reduce injurious effect in wounded area if 100% oxygen breathed in HBOT chamber with pressure more than 1 ATA (Gokce et al, 2008;
Inokuchi et al, 2009). During tissue repair and wound healing, oxygen demand and utilization rates are increased, and chronic hypoxia in a wound is associated with reduced or absent wound healing (Lam, 2017).
Daily HBOT supports an adequate oxygen supply to the site of injury to promote wound healing progression from the inflammatory phase to the proliferative phase. Hypoxia is a stimulus for angiogenesis. New blood vessel formation, is dependent on normal levels of oxygenation.
Hyperbaric oxygen therapy creates a gradient between tissues of low oxygenation in the center to higher concentrations at the periphery, thus modulate neovascularization (Lam, 2017; Hady). This condition will increase fibroblast and collagen production in wounded area. Increase daily exposure to HBOT also known to modulate the rate of osteoblast differentiation by increased alkaline phosphatase activity and expression of type I collagen and Runx-2 mRNA as parameter during the early stages of culture (Hady, 2015). HBOT in maxillary suture expansion has not been investigated yet
Research about HBOT in alveolar bone remodeling process during suture expansion has minimally studied before. Other research showed that the use of HBOT in 2,4 ATA in pressure area for 7 days is relatively better than 5 days in increasing tissue vascularization thus stimulates remodeling process. While the use of HBOT in 7 days and 10 days after inserting the orthodontic brackets showed that the use of HBOT 2,4 ATA in 10 days did not reflect significant increase as compared to the use of HBOT in 7 days (Ramadhani et al, 2014).
This research aims to analyzed balancing osteoclast/osteoblast in bone remodeling induced by giving HBOT 2,4 ATA given from day 8-14 to bone surface area during maxillary expansion.
MATERIALS AND METHODS
Design of this research is randomized post-test only control group. Ethical permission was issued from Ethics and scientific committee of experimental animal use at Faculty of dentistry Hang Tuah University 079/ KEPK/ III/2018.
This true experimental used eighteens male guinea pigs for sample with the criteria of: male, age of 3-4 months with body weight around 300-400
grams divided into 3 groups. Negative control group K(-), positive control was applied with maxillary expansion or K (+), the third group was applied with hyperbaric oxygen therapy (P). First, the guinea pig was acclimatized for 7 days. K(+) and P group was applied with helical spring to expand maxillary in day 3rd until 14th. Helical spring selected in this research is a slow maxillary expansion type. Before applying helical spring, rubber separator with tensile strength 0,29 gr/cm2 was used in left incisive until the second day to get diastema, then the third day rubber separators with tensile strength 0,48 gr/cm2 was replaced with helical spring. In P group, after day 8th based on previous research were placed into animal chamber for HBOT with 2,4 ATA pure oxygen (100%) for 90 minutes (3 X 30 minutes) with interval 5 minutes of breathing normal air (normobaric) until day 14th.
Image 1. Helical spring (slow maxillary expansion) was applied in maxilla
After HBOT for 14 days was applied for the assessed groups, the pressure of oxygen, Cavia cobaya were euthanized in lethal dose, and the maxilla were decapitated. Specimens of the maxilla were cut transversely and placed in 10% buffer formaline before made into slides, and then coloured using Hematoxyllin eosin colouration. Histological slide of osteoclast, osteoblast, and bone surface area were examined under light microscope in 40X magnification. Bone surface area were measured was the alveolar bone area between two central incisive.
This Research was conducted at Biochemistry laboratory Medical Faculty and Oral Biology Laboratory Hang Tuah University. Equipments used in this research are the Cavia cobaya cage, Cavia cobaya weighing scale, Waring Commercial Model HGBTWT, rubber separators, caliper, separating plier, maxillary expansion, pinset, cotton pellet, surgical scissors, small sized coil, animal chamber for HBOT.
Data obtained from the calculation number of in tension area were tabulated and were analyzed by regression test to know correlation between osteoclast, osteoblast, osteoclast osteoblast ratio and bone surface area during maxillary expansion.
RESULTS
Osteoclast number, osteoblast and bone surface area by giving HBOT 2,4 ATA given from day 8-14 as result of research groups are as shown in the picture below:
Image 2. Hematoxyllin Eosin colouration of osteoclast, osteoblast and bone surface area in K(-)
group, K(+) group, and P group with 40x magnification of during maxillary expansion.
Descriptive analysis from this research showed distribution and summary of data hence clarifying results presentation, and then regression tests were done using analytic statistic tests with significant level of 95% (p=0,05) using SPSS version 17 programme.
Research data in table 1 shows a linear increase from K(-) to P. Table shows that the HBOT increased osteoclast, osteoblast number, and osteoclast/osteoblast ratio in tension area during maxillary expansion.
Table 1. Data results of the means and standard deviations osteoclast treated by HBOT.
Group Mean (cells) Standard Deviation
K(-) 0.00 ±0.00
K(+) 0.15 ±0.16
P 1.71 ±0.34
Table 2. Data results of the means and standard deviations osteoblast treated by HBOT.
Group Mean (cells) Standard Deviation
K(-) 14.87 ±0.61
K(+) 20.23 ±0.91
P 37.95 ±1.85
Table 3. Data results of the means and standard deviations osteoclast/osteoblast ratio treated by
HBOT Grou
p
Mean of osteoclast/osteoblast
ratio
SD
K(-) 0.0 ±0.0
K(+) 0.01 ±0.01
P 0.05 ±0.01
Table 4. Data results of the means and standard deviations bone surface area treated by HBOT Group Mean of bone surface
area
SD
K(-) 0.66 ±0.18
K(+) 0.84 ±0.32
P 1.14 ±0.24
Image 4. Bar diagram of osteoclast, osteoblast, osteoclast/osteoblast ratio, bone surface area in
tension area during maxillary expansion
Before conducting hypothesis test, normality test for each group was conducted using Shapiro – Wilk test, as the samples were less than 50.Results from Shapiro – Wilk test showed that the data were distributed normally.
Tabel 4. Regression test HBOT treatment Group Significancy (R)
K(-), K(+), P 6,64
Tabel 5. Anova test HBOT treatment Group Significancy (R)
K(-), K(+), P .05
Regression test showed that there is strong positive correlation between osteoclast number, osteoblast number, and osteoclast/osteoblast ratio to bone surface area with coefficient of multiple determination R=6.64 in HBOT during maxillary expansion. Greater osteoclast number, osteoblast number, osteoclast/osteoblast ratio, and bone surface area is associated, so is the the higher of osteoclast number, osteoblast number and osteoclast/osteoblast ratio getting higher also and so that bone surface area.
osteoclas t
Osteoblas t
Bone surface
osteoclas t/osteobl ast ratio
K- 0 14,87 0,66 0
K+ 0,15 20,23 0,84 0,01 P 1,71 37,95 1,14 0,05 0
5 10 15 20 25 30 35 40
Anova test results signified that all groups in this research had significance difference between osteoclast number, osteoblast number, osteoclast/osteoblast ratio and bone surface area with HBOT during maxillary expansion.
DISCUSSION
Results from descriptive statistic tests showed that the highest means of osteoclast number, osteoblast number, osteoclast/ osteoblast ratio, and bone surface area is P group that treated with HBOT in 2,4 ATA. This was due to the fact that HBOT could increase neovascularization and angiogenesis that is closely related in bone formation process—in increasing osteoblastic activity (Sutomo, 2012).
Osteoblast plays a role in the synthesis of bone components, specifically the type I-collagen, proteoglycan and glycoprotein which includes osteonectin (Ramadhani, 2014).
When helical spring was treated in K(+) there is minimal new bone formed compare to K(-) and P.
Orthodontic forces are known to occlude periodontal ligament vessels on the pressure side of the dental root, decreasing the blood perfusion of the tissue.
This condition is accompanied by hypoxia, which is known to either affect cell proliferation or induce apoptosis, depending on the oxygen gradient. In recent years, considerable interest has arisen in the mechanisms whereby hypoxia and the hypoxia- inducible transcription factors, HIF-1α and HIF-2α, affect bone remodeling and bone pathologies (Knowles, 2015). Because upregulated tissue proliferation rates are often accompanied by angiogenesis, hypoxia may be assumed to fundamentally contribute to bone remodeling processes during orthodontic treatment (Niklas et al, 2013).
Acute exposure to hypoxia also increases the ability of mature osteoclasts to resorb bone (Knowles, 2015). Remodeling of the bone margins of the palatal suture was evident with different maturation stages of the newly-formed bone areas characterized by wide osteocyte lacunae (Pereira, 2017). Clearly, in the in vivo mechanism, monocytes and osteoclasts do not exist alone but are surrounded by osteoblasts, fibroblasts, and other cellular components of the bone microenvironment that will also be exposed to local hypoxia. Co-culture of monocytes with stromal cells including osteoblasts, fibroblasts, and cancer cells has revealed that hypoxia stimulates local production of pro- osteoclastogenic cytokines including RANKL vascular endothelial growth factor (VEGF), M-
CSF,27 insulin-like growth factor and growth differentiation factor as well as inhibiting production of osteoprotegerin (OPG), a soluble decoy receptor for RANKL that inhibits osteoclast formation and activity (Knowles, 2015).
Based our research, we also speculate that minimal new bone formed because of helical spring that we used here as slow maxillary expansion would have a less forced to make skeletal effect (Angilieri, 2013). helical spring approaches were able to make some changes, especially at dental level also transversal level changes involved both skeletal and dentoalveolar due to maxillary expansion (Pereira, 2017).
The ossification process during maxillary expansion begin from suture margins along with bone islands (bone masses of acellular tissue and inconsistently calcified tissue) in the middle of the sutural gap as we see in image 1 in K(+).
(Korbmacher, 2007; Angilieri, 2013). The formation of spicules during sutura maxillary expansion occurs in many places along the suture, with the number of spicules increasing with maturation (Angilieri, 2013) and forming many scalloped areas that are close to each other and separated in some areas by connective tissue. Simultaneous, interdigitation increases; then fusion occurs earlier in the posterior area of the suture, with progression of ossification taking place from posterior to anterior, with resorption of cortical bone in the sutural ends and formation of cancellous bone (Werbein, 2001;
Knaup, 2004; SunZ, 2004).
At the end of expansion newly-formed bone with osteoid matrix undergoing mineralization was evident on the bone margins and from within the center of the suture, as was the peculiar fishbone appearance of the trabecular bone. Moreover, newly- formed bone showed collagen fibers in a transversal orientation related to the suture long axis where a longitudinal orientation was observed. This orientation was suggested to be related to the response to mechanical forces (Hou, 2007). Once the midpalatal suture separation is obtained, the amount of expansion might affect the healing time of the bone that starts after the midpalatal separation and keeps going for several months after (Pereira, 2017).
P group was helical spring that given with HBOT showed that increasing osteoblast number and bone surface area than K(+) group. It is indicated that HBOT during maxillary expansion can accelerated new bone formation. It is marked with spongiosa bone replaced with compact bone.
Surprisingly, HBOT treatment also increase osteoclast number, so that osteoclast/ osteoblast ratio, but not that higher than osteoblast number. It is related to condition stage of hypoxic (Knowless, 2015). HBOT become increasingly resistant to RANKL-induced osteoclast formation. Effect of early-stage HBOT on mononuclear and multinuclear osteoclast formation, suggesting that the response varies according to the stage of precursor development—with monocytes further along the osteoclastic lineage being more resistant to the anti- osteoclastic effect of HBO than less committed cells (Al Hadi, 2015).
Maxillary expansion will cause deformation of the blood vessels and the irregularity of tissues around the tooth. As a result, there will be change in the cell metabolism due to the hypoxic condition and decreased nutrition levels. This would cause the local inflammation process. HBOT have mechanism to decrease inflammation (Ariffin, 2011).
During inflammation under hypoxia, can stimulate the production and activity of human osteoclasts derived from PBMCs, where there is effectively no stromal cell support, as well as from pure populations of CD14+ monocytes, suggests that the osteoclastogenic response to hypoxia is an intrinsic property of this cell lineage (Al Hadi, 2015).
Hipoxia also showed increase of VEGF mRNA, and also to that the RNA's half-life was extended. This effect is translated by the hypoxia sensitive transcription factor HIF-1. HBOT could increase the levels of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are the most extensively angiogenic factors which causes neovascularization and angiogenesis that is closely related in increasing osteoblastic activity and vital for new bone formation process (Sutomo, 2012).
Decrease of proinflammation mediators which causes the hampered formation and activity of osteoblast proliferates dan differentiates into mature osteoblasts hence the number of osteoblast increases bone remodeling new bone formation and the bone density would increase as well. VEGF164 activates the PI3K/AKT pathway in osteoblasts and induces a stabilization and signal transduction by the main component of the Wnt signaling pathway, β-catenin.
Overexpression of VEGF164 isoform leads to osteosclerosis, highly increased bone formation, accompanied by an intensified osteoblast differentiation resulting in bone overgrowth and altered bone morphology. This indicates a need for a tight angiogenesis control during bone development (Maes, 2004).
Regression test showed that there is strong positive correlation between osteoclast number and osteoblast number, osteoclast/osteoblast ratio to bone surface area with coefficient of multiple determination R=6.64 in HBOT during maxillary expansion. Greater osteoclast number and bone surface area is associated, so is the osteoblast number and bone surface area have sinergystic effect. Both physical and biochemical, bone cells are connected while many factors involved in the conversation between endothelial cells (EC), osteoclasts and osteoblasts (OB) during both bone formation and repair such as BMP-2, OP-1 (Carano, 2003).
Previous research showed that HBOT resulted in a significantly increased new bone formation and angiogenesis compared to the sole treatment with autologous bone grafting that proved that HBOT have role in both angiogenesis and bone remodelling (Grassman, 2015). Over about 30 treatments new vessel growth infiltrated the wound and achieved pO₂'s of about 85% of control tissue. The mechanisms of HBOT action have been proposed:
(1). HBOT transports oxygen to bodily sites where vascularization is poor or absent, such as in poorly healing wounds. This proposed mechanism of HBOT action relates to the physical relationship between pressure and gas concentration in a liquid It is known that 2.4 ATA of pure oxygen dissolves a substantial amount of oxygen in blood plasma; (2). Cyclic periods of hyperbaric oxygen and normoxic oxygen create a stress response by repeatedly increasing and decreasing the number of reactive oxygen species (ROS) in the tissues. ROS influence the signal transduction pathways of multiple growth factors, including those implicated in propagating angiogenesis (Van Neck, 2017).
Anova test results signified that all groups in this research had significance difference between osteoblast number, osteoblast number, osteoclast/osteoblast ratio and bone surface area with HBOT during maxillary expansion. HBOT become increasingly resistant to RANKL-induced osteoclast formation (Al Hadi, 2015), the other side, also effect to stimulate the proliferation and differentiation of human osteoblasts in vitro. Also HBO enhanced biomineralization with an increase in bone nodule formation, calcium deposition, and alkaline phosphatase activity (Wu, 2006) and also expression of collagen type 1 and Runx-2 (Hadi, 2015). HBOT significantly promoted osteoblast proliferation and cell cycle progression after 3 days of treatment.
HBOT stimulated significantly increased mRNA expression of fibroblast growth factor (FGF)-2 as well as protein expression levels of Akt, p70S6K,