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Comparative analysis of bone remodelling markers In anterior and posterior segment after micro-osteoperforation- A GCF study
Jose Sunny1, K Nillan Shetty 2, Gautham Hegde3, Shetty Suhani Sudhakar4*, Sherine Sue Mathew5
1Al Azhar dental college , Thodupuzha, Kerala, India
2, 3A.J.Institute of dental sciences, Mangalore, Karnataka, India
4 Srinivas Institute of Dental sciences, Mangalore, Karnataka, India
5 Private practitioner, Ernakulam, Kerala, India
*[email protected] ABSTRACT
OBJECTIVES– The objectives of this study is to evaluate the difference in level of bone remodeling markers in GCF of anterior and posterior segments after micro-osteoperforations.
MATERIALSANDMETHOD- GCF samples of 15 patients undergoing micro-osteoperforations as a part of ongoing orthodontic treatment was used, samples were collected from the buccal crevices of canine and second premolar on 14th day after micro-osteoperforation, Samples for baseline was collected before micro-osteoperforation from the contra lateral side, Micro osteoperforation was performed using a mini implant of length 6mm on one side of the arch. For collection of gingival crevicular fluid, Perio paper was held within the gingival sulcus 1 mm below the gingival margin for 30 seconds. The strips with the collected sample were transferred to the phosphate buffer saline and the samples were stored at -700 C
RESULTS- There is statistically significant relation between the levels of alkaline phosphatase in both anterior and posterior segments after micro-osteoperforation (p value <0.002) whereas there was significant co-relation only on the anterior segment when acid phosphatase was evaluated( p <0.001) and in the posterior segment there was no statistically significant value was obtained.
CONCLUSION- After micro-osteoperforation, there was significant increase in the level of acid phosphatase noticed in the anterior segment whereas there was significant increase in the level of alkaline phosphatase noticed in both the anterior and posterior segment. The results obtained showed that there is increased remodeling occurring in the anterior segment than the posterior segment
KEYWORDS: GCF, MOP, ACP, ALP
Introduction
Orthodontic treatment is usually considered boon to the patient, however one of the drawback perceived by many is the prolonged treatment time, leading patients especially adults to seek out other less than optimal treatment options like veneers, crowns and bridges1.Therefore, methods that decrease the treatment duration without compromising the outcome is the need of the hour.
To accelerate the tooth movement local and systemic pharmaceutical administration, physical and mechanical stimulation have been utilised.2The practicality of these methods on daily clinical practice,their side effects and validation of results are awaited.3
The osteoclast activity controls the bone resorption rate which in turn controls the rate of tooth movement4-5.So factors affecting osteolclastic activity would in turn affect the rate of tooth movement. However, a simple non-invasive method is required for achieving these possibilities.
In recent times,a few components of gingival crevicular fluid (GCF) have been shown to be diagnostic biomarkers of active tissue destruction in periodontal disease which might also serve as diagnostic markers of biologic responses in orthodontic tooth movement.6
Various studies suggested that the mechanical stimulants like orthodontic tooth movement may induce some subsequent inflammatory responses in periodontal tissue.7 The cells can release enough amounts of chemical mediators into GCF, therefore the amount of these substances may increase during orthodontic tooth movement in GCF 8
Considering many of biologic bone markers, alkaline phosphatase (ALP) and acid phosphatase (ACP) have been examined as bone turn over markers in orthodontic tooth movement.6,9-10
ALP, a glycoprotein is involved in mineral formation in tissue like bone and cementum.11 Robinson (1923) was first in suggesting that the ALP is important in the mineralization of bone and calcifying cartilages.12 The enzyme is thought to release phosphate ions from organic phosphate esters as a result of which supersaturation would occur, leading to the precipitation of calcium phosphate salts.11 This enzyme is produced by neutrophils at GCF, although it is also produced by variety of cells including fibroblasts, osteoblasts, and osteoclasts 13
ACP is also an enzyme thought to be involved in processes of mineral deformation in tissues like bone. Resorbing cells, such as osteoclasts and macrophages, have been shown to have high ACP activities 10,14
Keeling et al. (1993) examined tartrate-resistance acid phosphatase (TRAP) changes in serum and alveolar bone during orthodontic tooth movement in 288 adult male rats. The authors reported that enzyme activities increased during orthodontic tooth movement. In addition, high levels of ACP were detected on first day in serum and on third day in bone, whereas the peak of ALP activity in bone has occurred on seventh day10.
Parinettiet alhave examined ALP activity in gingival crevicular fluid during orthodontic tooth movement and demonstrated that the ALP activity was significantly higher in tension sites rather than compression sites of teeth undergoing orthodontic forces9.
Studies have shown an increase in the activity of inflammatory markers such as chemokines and cytokines in response to orthodontic forces.15-16 Chemokines play an important role in the recruitment of osteoclast precursor cells, and cytokines, directly or indirectly, through the prostaglandin E2 pathway and the RANK/ RANKL pathway, lead the differentiation of osteoclasts from their precursor cells into mature osteoclasts.17-18
Therefore, increasing the expression of these factors should accelerate tooth movement. Various animal studies have shown that performing micro-osteoperforations (MOPs) on alveolar bone during orthodontic tooth movement can stimulate the expression of these inflammatory markers, leading to increases in osteoclast activity and the rate of tooth movement.1Objective of this study is to estimate the level of acid phosphatase and alkaline phosphatase in GCF in anterior and posterior segment after micro-osteoperforation in humans
Materials and methods
GCF samples of 15 patients undergoing micro-osteoperforations as a part of ongoing orthodontic treatment in outpatient department of orthodontics and dentofacial orthopedics.
Inclusion criteria
•Age group 18 – 45 years.
•First premolar extraction.
•Healthy gingival and periodontal condition.
Exclusion criteria
•Periodontal diseases.
•Systemic diseases.
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•Smoking.
•Previous orthodontic treatment /Orthognathic surgery.
Based on the inclusion criteria, 15 patients requiring retraction will be selected and informed consent obtained from the patients prior to the procedure. Complete levelling and aligning of the arches will be done using sequential wires in 0.022’ slot MBT brackets.
If supragingival plaque was present it will be removed and patient is made to rinse with Betadine mouthwash twice. Teeth is gently sprayed with an air spray and properly isolated with cotton rolls. Sample will be taken from the buccal crevices of canine and second premolar (Figure 1) on 14th day after micro-osteoperforation (MOP), Samples for baseline will be collected before micro-osteoperforation from the contra lateral side. Microosteoperforation is performed using a mini implant (Dentos) of length 6mm on one side of the arch at the extraction site . Two perforations of depth 6mm was given at the extraction site (Figure 2)
Figure 1- Perio paper in buccal crevices of canine and 2nd premolar
Figure 2- perforations in extraction site
For collection of GCF Perio paper (Perio paper, oraflow, NY) will be held within the gingival sulcus 1 mm below the gingival margin for 30 seconds. Strips contaminated by blood or saliva would be discarded. The strips with the collected sample will be transferred to the phosphate buffer saline in an Eppendorf tube. samples are stored at -700 C till analysis is done (Figure 4) using alkaline and acid phosphatase reagent (Figure 5)
Figure 4- Sample storage at -70°C
Figure 5- Alkaline phosphatase and acid phosphatase reagent
RESULTS
Since the observed values of alkaline phosphatase activity in Gingival Crevicular Fluid (ALP in GCF) in mesial and distal sites was close to the expected values, it was assumed that the said activity follows normal distribution. Comparison between the sample taken before and after MOP was done and the values that were obtained are given in Table 1.
TABLE 1: MEAN DISTRIBUTION OF THE ALKALINE PHOSPHATASE (BEFORE AND AFTER)
Alkaline phosphatase N Minimum Maximum Mean Std. Deviation
Before
Anterior 15 10.35 37.53 22.9547 7.31884
Posterior 15 12.12 31.67 21.8487 5.32206
After Anterior 15 16.62 67.87 36.3640 13.24781
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Posterior 15 15.18 60.89 33.6622 13.30422
A mean of 22.954 U/L was obtained with a maximum value of 37.53 U/L and minimum value of 10.35U/L in the anterior segment and a mean of 21.848 U/L was obtained with a maximum value of 31.67 U/L and a minimum of 12.12 U/L for the posterior segment was obtained.
After MOP there was significant increase in the level of ALP noticed in both the anterior and posterior segment as depicted in Graph 1.The highest value was obtained at the anterior segment which was 36.364 U/L and this denotes higher incidence of ALP in the anterior segment than the posterior segment.
The observed values of acid phosphatase activity in Gingival Crevicular Fluid (ACP in GCF) was close to the expected values, the activity of ACP follows normal distribution.Comparison between the sample taken before and after MOP. The values that were obtained are given in Table 2.
Table 2- Mean distribution of acid phosphatase ( Before and after micro-osteoperforation)
Acid phosphatase N Minimum Maximum Mean Std.
Deviation
Before
Anterior 15 .55 1.56 .9605 .32488
Posterior 15 .39 1.70 .7695 .37915
After
Anterior 15 .84 3.44 1.5366 .64534
Posterior 15 .15 1.28 .8575 .27708
A mean of 0.954 U/L was obtained with a maximum value of 1.56 U/L and minimum value of 0.55U/L in the anterior segment and a mean of .7695 U/L was obtained with a maximum value of 1.7 U/L and a minimum of 0.39 U/L for the posterior segment was obtained.
After MOP there was significant increase in the level of ACP noticed in the anterior segment and posterior segment there was not much change, As depicted in Graph 1. The highest value was obtained at the anterior segment which was 1.536 U/L and this denotes higher incidence of ACP in the anterior segment than the posterior segment indicating more bone remodelling in the anterior segment than the posterior during retraction.
A comparison of both ACP and ALP was done using a paired sample t test depicted by Table 3.
TABLE 3: COMPARISON OF THE ALKALINE PHOSPHATISE AND ACID PHOSPHATISE (BEFORE AND AFTER) USING PAIRED SAMPLE t TEST
Mean difference t value p value Alkaline
phosphatase
Anterior -13.40 -4.62 0.00*
Posterior -11.81 -3.90 0.002*
Acid phosphatase Anterior -0.57 -3.99 0.001*
Posterior -0.08 -0.75 0.46
There is stastically significant relation between the levels of ALP in both anterior and posterior segments after MOP. (p value <0.002) whereas there was significant co relation only on the anterior segment when ACP was evaluated.( p <0.001) and in the posterior segment there was no stastically significant value was obtained.
The results obtained showed that there is increased remodelling occurring in the anterior segment than the posterior segment, corelating with the retraction of the anterior segment which takes up more extraction space than the posterior segment i.e anchorage is preserved.
So the bone remodelling markers are a good method to assess the treatment progress.
Discussion
Orthodontic tooth movement produces remodelling of bone with deposition in tension sites and resorption in pressure sites 39-40. Various animal studies have shown that a sequence characterized by periods of activation, resorption, reversal, and formation has been described as occurring in both tension and compression tooth sites during orthodontic tooth movement41
In orthodontics, mechanical stress appears to bring about biochemical and structural responses in a variety of cell types in vivo and in vitro 9.The early phase of orthodontic tooth movement involves an acute inflammatory response, characterized by periodontal vasodilation and migration of leukocytes out of periodontal ligament capillaries47.The mechanism of bone resorption might also be related to the release of inflammatory mediators that can be detected in the gingival crevicular fluid48.Proper oral hygiene has been emphasized by thorough oral hygiene practices and periodic examination and scaling to control the gingival inflammation in the present study.
Several different approaches are adopted for the collection of GCF. A method which is less disturbing to the crevicular epithelium and facilitates more rapid measurements is the placement of filter paper strips in the gingival crevice 49-50 was adopted over using micro capillary tubes 51.
Basic bone biology research, animal studies and controlled clinical trials have demonstrated the safety and efficacy of the MOP treatment. Various results of such studies have demonstrated that micro-osteoperforation decreases orthodontic treatment in combination with any type of orthodontic force. Reducing treatment time for patients has been a goal due to patient and orthodontist demand and as such reducing the number of these multiple office visits will save both time and moneyThe present study shows increase bone remodeling in the anterior segment than the posterior segment on applying the orthodontic force for retraction as well as using micro
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osteoperforation to accelerate the tooth movement by incorporation the RAP mechanism described by frost.
ALP activity in orthodontic tooth movement is of significance as it is an essential enzyme for bone deposition 42and thus a reliable marker of osteoblastic activity 43.Alkaline phosphatase (ALP) is an enzyme of the hydrolase class of enzymes and it acts in an alkaline medium. It is age dependent and is found in high concentrations in bones, liver, and biliary tract epithelium. Any change in alkaline phosphatase level and activity is seen in a variety of physiological and pathological events such as bone development, bone-related diseases like Paget’s disease, rickets, osteomalacia, liver disease, inflammatory bowel disease, hyperparathyroidism, post parathyroidectomy, gestation and in drug toxicity. Hence, selection of patients for the study was such that they exhibit good general health.44
After inserting an orthodontic force, an early phase (3-5 days) of non-specific bone resorption activity is followed by an early (5-7 days) and a late phase (7-14 days) of bone deposition.9In the current study, ALP activity was measured on the 14th day in both mesial and distal of the tooth measured which is supported by the earlier hypothesis that increased osteoblastic activity could take place in both tension and compression sites in that time period in the alveolar bone.32 Investigations by Christenson also reported bone formation appeared to begin after osteoclastic resorption phase that could last from 10 days to 3 weeks.45The results obtained in the present study, which are in agreement with other studies, suggest that the increase in ALP activity in GCF might be related to dental site bone remodeling following orthodontic forces46
ACP also is seen as a biologic marker of bone destruction.43Acid Phosphatase (ACP) is an enzyme of the Hydrolase class of enzymes and acts in an acidic medium and is widely distributed and found in high concentrations in the liver, RBC’s and the prostate. Increased levels of the prostatic fraction are associated with prostatic carcinomas. Increased levels of the non- prostatic fraction are associated with liver diseases, hyperparathyroidism and Paget’s disease. Hence, to avoid any false reading the criteria of selection of patients for the study was that they exhibit good general health.
Data from present study, as well as other studies, have shown that in the early phase of tooth movement, the ACP activity is greater than later phase that follows by increasing in the ALP activity and decreasing in the ACP activity. Investigation by Farahani M and Puneet showed a peaking of ACP values towards the 14th day20-21. The maximum ACP activity was reported between 3 and 6 weeks in Insoftet al. study. The results obtained in the present study, suggest that the increase in ACP activity in GCF might be related to dental site bone deposition.
This study showed that monitoring ALP and ACP activity at different sites of teeth undergoing orthodontic tooth forces could help the clinicians to better understand the local biological process. Longitudinal studies of orthodontic patients can increase our knowledge about possible positive effect of orthodontic tooth movement and bone reconstruction on applying the orthodontic forces.
Conclusion
From the values of this study it can be concluded that estimation of bone remodeling markers in GCF that is ALP and ACP levels serve as an indicator of the rate of remodeling of the tissues during tooth movement.
This can serve as tool to customize the orthodontic force that has to be applied in individual cases. Additionally, the understanding of the GCF bone turnover markers could also be used to assess the treatment progress.
Acknowledgement Nil
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