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 Table of Contents  
ORIGINAL ARTICLE
Year : 2015  |  Volume : 49  |  Issue : 1  |  Page : 10-14

Assessment of acid phosphatase and alkaline phosphatase in gingival crevicular fluid in growing and adult orthodontic patients: An in vivo study


1 Senior Lecturer, Department of Orthodontics and Dentofacial Orthopedics, M.R. Ambedkar Dental College and Hospital, Bangalore, India
2 Professor and HOD, SDM College of Dental Sciences and Hospital, Dharwad, India
3 Professor, P.M.N.M Dental College and Hospital, Bagalkot, Bangalore, Karnataka, India

Date of Submission06-Nov-2014
Date of Acceptance07-Apr-2015
Date of Web Publication12-Jun-2015

Correspondence Address:
Smitha V Shetty
Department of Orthodontics and Dentofacial Orthopedics, M.R. Ambedkar Dental College and Hospital, Bangalore, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0301-5742.158627

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  Abstract 

Introduction: Orthodontic treatment has been widely accepted among adults in the present years. It has been postulated that the tooth movement is faster in children when compared to the adults. This could be assessed more accurately by assessment of gene markers responsible for bone turnover in the gingival crevicular fluid (GCF). The purpose of this study was to assess the association of levels of acid and alkaline phosphatase activities in GCF with age during the tooth movement. Materials and Methods: A group of 14 growing and adult patients requiring fixed orthodontic therapy were selected. The maxillary canines were retracted in both the groups using coil springs. GCF samples were collected before the spring activation and subsequent sampling was done after the activation up to 6 th week. The levels of alkaline and acid phosphatase were then compared. Results: On comparison, the alkaline phosphatase levels were much higher in children at baseline and remained elevated suggesting increased osteoblastic activity. The levels of acid phosphatase were higher in the children at all-time intervals, indicating increase in the osteoclastic activity. Conclusion: Increase in enzymatic activity in children during the tooth movement as compared to adults partially supports the hypothesis of faster tooth movement in children.

Keywords: Acid phosphatase, adults, alkaline phosphatase, children, gingival crevicular fluid, orthodontic tooth movement


How to cite this article:
Shetty SV, Patil AK, Ganeshkar SV. Assessment of acid phosphatase and alkaline phosphatase in gingival crevicular fluid in growing and adult orthodontic patients: An in vivo study. J Indian Orthod Soc 2015;49:10-4

How to cite this URL:
Shetty SV, Patil AK, Ganeshkar SV. Assessment of acid phosphatase and alkaline phosphatase in gingival crevicular fluid in growing and adult orthodontic patients: An in vivo study. J Indian Orthod Soc [serial online] 2015 [cited 2019 Jun 16];49:10-4. Available from: http://www.jios.in/text.asp?2015/49/1/10/158627


  Introduction Top


In orthodontics, stresses applied to a tooth during the tooth movement evoke various biochemical and microstructural responses demonstrating the underlying bone remodeling. Due to the mechanical stresses, extensive macroscopic and microscopic changes are seen in the dental and paradental tissues. [1],[2] Bone remodeling that occurs during orthodontic tooth movement is a biologic process involving an acute inflammatory response in periodontal tissues in order to understand and develop the biological logarithms for stimulating tooth movement in bone, the underlying complex mechanisms of bone resorption and apposition should be understood in detail. This complex and active process of bone turnover involves the bone formation by osteoblasts and resorption by osteoclasts. [3] The osteoclasts have high acid phosphatase activity and the osteoblasts have alkaline phosphatase activity. [4],[5],[6] Monitoring the phosphatase activities in the gingival crevicular fluid (GCF) could be suggestive of the tissue changes occurring during the orthodontic tooth movement. [7],[8],[9]

Recent advances in clinical techniques and changes in patient's awareness of malocclusion have resulted in increased demand for orthodontic treatment among adults. The difference in the anatomic structure and tissue response to orthodontic tooth movement at different age groups must be considered. It has been found that the amount of tooth movement is significantly greater in younger individuals than in adults. [10],[11] However, the effect of age on the rate of tooth movement is still unclear.

Most of the studies done earlier, to estimate the factors responsible for bone turnover have been conducted by invasive techniques and by sacrificing the experimental animal. [12],[13] Assessments of gene markers in GCF have been the forefront research topic in the same context. An understanding of the biochemical and molecular mechanisms that enable bone cells to adapt to changes in their mechanical environment is essential for the practice of clinical orthodontics. Furthermore, by monitoring the bone turnover of a patient and relating it to the patient's age, an optimal force can be applied, which caters to the patient's need. There has been no study done to analyze the influence of age on the levels of acid phosphatase and alkaline phosphatase in GCF, which may be the direct evidence to understand the basic difference in bone turnover in children and in adults. This study monitors acid and alkaline phosphatase activities in GCF and correlates it with different age groups making it possible to biologically monitor and predict the outcome of orthodontic forces based on the individual tissue response.


  Materials and Methods Top


The study group of 14 growing patients (mean age group of 11 ± 4 years) and 14 adults (mean age group 25 ± 5 years) requiring fixed orthodontic therapy with bilateral maxillary first premolar extractions were selected. All patients were checked for periodontal status, and those with a history of systemic diseases or history of intake of any medication within the past 3 months were excluded from the study. The research was carried out in compliance with the Declaration of Helsinki after obtaining ethical approval from the Institutional Review Board, SDM College of Dental Sciences and Hospital, Dharwad, India.

After obtaining the informed consent, fixed orthodontic therapy was started in all the patients. After the stage of leveling and aligning and adequate bite opening, the experimental canines were retracted individually in both children and adults using Nickel Titanium coil spring (Orthoforce G4, G, and H wire company) on a 0.018 stainless steel round wire (Special plus, A J Wilcock, Whittle sea, Australia) delivering a force of 150 gm. The force was measured using a Dontrix gauge. However, anchorage was reinforced using a transpalatal arch on the molars.

Gingival crevicular fluid collection

Supra gingival plaque was removed from the maxillary right canine (experimental tooth); the region was flushed with water and gently dried with air. The isolation of the teeth was obtained using a self-retaining retractor, suction, and cotton rolls.

From each test site, a standardized volume of 2 μl was collected using the calibration on white color-coded 1-5 μl calibrated volumetric microcapillary pipettes (Sigma-Aldrich Chemical Company, St. Louis, MO, USA) using an unstimulated extracrevicular approach.

The micropipette was introduced at the entrance of the gingival sulcus and a standardized volume of 2 μl of GCF was collected from the distal side of maxillary canine (T 0 ) before the retraction spring activation [Figure 1].
Figure 1: Gingival crevicular fluid collection using micropipettes at baseline (T0)

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Subsequent sampling was done after the activation of the appliance using Dontrix gauge, that is, at day 7 (T 1 ), day 21 (T 2 ), and day 42 (T 3 ) [Figure 2]. The GCF was then diluted in phosphate buffer saline solution and stored at − 20°C until biochemically assayed. The biochemical assay was carried out for all the samples using semi-automated biochemical analyzer making use of commercially available kits.
Figure 2: Subsequent sampling of gingival crevicular fluid using micropipettes (T1, T2, T3)

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Statistical analysis

Significant changes in acid and alkaline phosphates levels between the different treatment periods were evaluated by using paired t-test. The levels of acid and alkaline phosphates in children and the adult groups were compared using unpaired t-test.


  Result Interpretation Top


Alkaline phosphatase

When the levels of alkaline phosphatase in the GCF at baseline (T 0 ) were compared between the adult and children group [Table 1], there was no statistical significance between the groups. At the 1 st week (T 1 ), there was an increase in both the groups (P = 0.0001), with significantly increased levels in children as compared to adults. However, at the 3 rd week (T 2 ), the levels of alkaline phosphatase remained elevated significantly (P = 0.0001) in children as compared to the adult. At the end of 6 th week (T 3 ), the levels still remained elevated in children group as compared to adult group, significantly (P = 0.0006).
Table 1: Intragroup and intergroup differences in alkaline phosphatase levels (IU/L)


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Acid phosphatase

When the levels of acid phosphatase in the GCF at baseline (T 0 ) and after 1 st week were same between the adult and children group, there was no statistical significance [Table 2]. However, at the 3 rd week (T 2 ), the levels of acid phosphatase showed significant increase (P = 0.0000) in the children group as compared to the adult. At the 6 th week, the increase in the levels of acid phosphatase were maintained in both the groups similar to 3 rd week (T 2 ), but was significantly higher (P = 0.0000) in children group as compared to the adult group.
Table 2: Intragroup and intergroup differences in ACP levels (IU/L)


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  Discussion Top


Bone is a remarkably labile tissue which is being constantly remodeled throughout life. The much proved and believed hypotheses that prolonged pressure on teeth results in remodeling of the alveolar bone, which consists of bone deposition and resorption is still the major platform for further research. [1],[2]

Molecules which are biologically active like phosphatases are secreted by the cells of the bone matrix at certain stipulated times to regulate the ongoing remodeling processes. [3] These biochemical markers are expressed as an exudate. GCF is one such exudates that represents a powerful, noninvasive vehicle, since it contains an array of biochemical and cellular factors which feature as biomarkers of the state of the periodontium. [14],[15] Thus, monitoring and analyzing the biochemical activities of the alveolar bone in the GCF could be suggestive of the tissue changes occurring during orthodontic tooth movement. [16],[17] This analysis could give us a clear picture of the underlying bone remodeling. The bone remodeling process can show timely changes in the biochemical markers, especially phosphatase activities during the various tooth movements. [17],[18],[19],[20],[21],[22],[23],[24],[25]

Over the past few decades, a tremendous increase in the demand for adult orthodontic therapy is seen. In the field of medicine, tremendous research have been done to differentiate bone turnover rate in children and adults. [26] However, our knowledge on the efficiency of adult tooth movement is still rather incomplete. Although it is reported that tooth movement occurs to a greater extent and at higher rates in younger individuals than in adults, there is no conclusive scientific evidence for the same. [27],[28],[29],[30],[31] Thus, this study was undertaken as an attempt to fill up these lacunae by using the present day advanced molecular research. In our study, we assessed and correlated two bone turnover biomarkers, acid, and alkaline phosphatase, in GCF. The biomarkers were then again compared between different age groups, hence making it possible for the orthodontist to biologically monitor and predict the outcome of the treatment based on the individual's tissue response to the orthodontic forces.

The duration of this study was 42 days (6 weeks) and the time interval of collection for GCF was programmed so as to identify and understand the enzymatic changes occurring during phases of orthodontic tooth movement.

Comparison of differences in the levels of acid and alkaline phosphatase in adults and children

Alkaline phosphatase


In this study, the levels of alkaline phosphatase in GCF were almost similar in both the groups at the baseline (T0). However, 1-week after the active canine retraction using NiTi coil springs (T1), there was a significant (P = 0.005) increase in the levels of alkaline phosphatase in GCF in both the groups, with statistically higher levels in children (P = 0.0001) as compared to the adults. This was coincident with the findings in the previous studies, which also showed an increase in the enzyme activity during the 1 st week of active orthodontic tooth movement. [3],[4],[10],[25] This increase in the enzyme activity might be explained by the fact that there is chemoattraction of osteoblasts or their precursors to the site of bone formation or to the site of inflammation during orthodontic tooth movement. The levels of alkaline phosphatase, however, remained constant at the end of the 3 rd week (T2) in both the groups. This constant value can be pointers of the continuity of the second phase orthodontic tooth movement. At the end of the 6 th week (T3), the enzyme activity showed a steep fall in the adult group reaching almost to the baseline value. In the children group, the enzyme activity continued to be higher as compared to the adults (P = 0.0006) even at the end of 6 th week suggesting prolonged bone depository activity of the osteoblasts in children.

In case of children, the reversal phase of orthodontic tooth movement by the osteoblasts to fill up the lacunae created during the resorption phase is prolonged. The prolonged increase in enzyme activity shows that there is simultaneous bone deposition occurring along with bone resorption even after 3 weeks of force application. This could partially explain the hypothesis of faster tooth movement in children as compared to the adults.

Acid phosphatase

The levels of acid phosphatases in the GCF at baseline (T0) in both the adult group and children group were almost same. There was no significant increase in the levels of acid phosphatase in both the groups at the end of the 1 st week (T1). This could be attributed to the recruitment of osteoclasts to the sites of tension after 5-8 days of orthodontic force application. Some of the past literature on biology of tooth movement has shown an early period characterized by a preponderance of bone resorption followed by a later period when bone formation is primary. [2],[4] The acid phosphatase levels significantly increased by the 3 rd week (T2) in both the groups: In adult group (P = 0.0001) and in children group (P = 0.0000) as compared with the baseline (T0). This increase could describe the second phase of orthodontic tooth movement, where there is a removal of the necrotic tissue and bone resorption from adjacent marrow spaces by osteoclast. There is a positive relation to the number of active osteoclasts and the rate of tooth movement. At the 6 th week (T3), the increase in the levels of acid phosphatase was maintained in both the groups similar to the 3 rd week (T2), but still it was significantly higher (P = 0.0000) in children group as compared to the adult group. This shows that the osteoclastic activity is increased in both the groups after 3 rd week but in children the levels have reached a higher value, indicating increased responsiveness of osteoclasts at the site of compression and frontal resorption during active tooth movement in children.

Both, alkaline and acid phosphatase levels were more in children as compared to the adults at any time interval studied. This suggests that there is an increased activity of the main bone remodeling cells, namely osteoblasts and osteoclasts, during the orthodontic tooth movement in children. This data are consistent with the previous studies on the effect of age on orthodontic tooth movement. [31],[32],[33] Changes in the proliferative activity of the remodeling cells are considered pertinent to the biologic phenomena associated with aging. Proliferative activities of the various cells in the periodontal tissue generally decrease with aging.

In younger age groups, the periodontal membrane is rich in cellular components, the alveolar bone crest is lined with uncalicified osteoid tissue, and the cancellous areas are filled with loose fibrous tissues, which is not generally the case in adults. [34] In adults, the decreased rates of bone turnover may be related to weak cellular response to the applied orthodontic forces. Furthermore, the bone density is lesser in younger individuals compared with the adults. [26] Bone remodeling during the tooth movement may also be affected by cell kinetics, several systemic, and local factors, neurotransmitters, and piezoelectricity, etc., Clearly, there may be multiple factors that could affect the rate of tooth movement in different age groups.

While designing a good diagnostic study, a major challenge is that there must be an accurate definition of the variable of outcome and the result must be quantified. In our study, the experimental canines have been moved bodily with a predetermined constant force, hence more the reliability of the results. Our results also confirms the differences in the proliferative activity of the periodontal ligament cells between young and adult groups during the different phases of tooth movement, suggesting that aging changes particularly affect initial cellular reaction in the periodontium subjected to external stimuli. Thus, it can be concluded that bone turnover can be monitored through the expression of alkaline phosphatase and acid phosphatase in GCF during the orthodontic tooth movement in different age groups.


  Conclusion Top


  • The levels of alkaline phosphatase increased in both the groups from the 1 st week and remained elevated up to the 3 rd week. This can be the pointer of the continuous prevalence of bone deposition. There was a significant decrease in the levels during the 6 th week in the adult group. However, in children the levels remained elevated suggesting prolonged bone depository activity of the osteoblasts during the postlag phase. Hence, faster tooth movement in children
  • The significant increase in the levels of acid phosphatase in children as compared to the adults during all the time periods indicates increased responsiveness of osteoclasts at the site of compression and frontal resorption in children
  • Thus, the results confirm the hypothesis of faster tooth movement in children as compared to the adults due to the increased acid and alkaline phosphatase activity in GCF reflecting an increased rate of bone remodeling in children.


 
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    Figures

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    Tables

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