Enzymatic evaluation of gingival crevicular fluid in cleft palate patients during orthodontic treatment: A clinico-biochemical study.

BACKGROUND: Therapeutic goal in patients with cleft lip and palate is esthetics and long-term health of the stomatognathic system. Patients with cleft lip and palate routinely require extensive and prolonged orthodontic treatment. The osseous structures are absent or poorly developed in the osseous clefts and may be traumatized in the course of orthodontic therapy; hence require constant monitoring during orthodontic treatment. The aim of the study was to evaluate the tissue response of cleft palate patients by quantitative analysis of enzyme activity during orthodontic treatment and assess any difference in the tissue response with that of noncleft patients undergoing orthodontic treatment.
MATERIALS AND METHODS: 20 patients requiring orthodontic treatment agedbetween 15 to 25 years were included to participate in the studyof which ten were cleft palate patients (group I) and ten noncleft patients (group II). The GCF samples were collected at incisor and molar sites during orthodontic treatment on days as per the study design in both the groups. The GCF enzymatic levels were estimated and compared.
RESULTS: Both groups showed significant increased enzyme activity at the incisor site compared to molar site corresponding to the phases of tooth movement.
CONCLUSION: There was significant difference in enzyme activity between the incisor adjacent to the cleft site and molar site. There was no difference in the tissue response between cleft palate patients and noncleft patients during orthodontic treatment.

INTRODUCTION

Cleft lip and palate (CLAP) is one of the most common of all congenital malformations of the face. It is prevalent among all races irrespective of sex. The involvement of the palate in CLAP is due to the absence of fusion of the palatal processes.[1] These birth defects may be associated with different syndromes such as trisomia 13 or the plateau- or Pierre–Robin syndrome.[2]

During orthodontic treatment, periodontal tissues respond rapidly to mechanical stress with consequent metabolic changes that allow tooth movement. One way of evaluating these changes is by analysis of the gingival crevicular fluid (GCF) composition. This non-invasive and simple method has been used to investigate the cellular response of the underlying periodontal ligament (PDL) and bone during orthodontic treatment.[3] Enzyme alkaline phosphatase (ALP) is commonly associated with bone metabolism with osteoblasts showing high alkaline phosphatase.[4] It is of particular interest in bone conditions associated with increased osteoblastic activity such as osteitis deformans where it rises 10-25-folds. Transient increase in activity is also observed during healing of bone fractures and physiologic bone growth.[5] Acid and alkaline phosphatases are released by injured, damaged or dead cells into extracellular tissue fluid. As a result of orthodontic force application, these enzymes produced in the periodontium diffuse into the GCF. An experimental study in rats suggested that phosphatase activities reflect bone turnover in orthodontically treated tissues.[6] Aspartate aminotransferase (AST) is a soluble enzyme that is normally confined to the cytoplasm of cells, but is released to the extra cellular environment upon cell death.[7] The presence of AST enzyme in GCF has been demonstrated and several studies have observed that the levels of GCF AST activity may reflect the magnitude of periodontal tissue destruction in periodontitis.[89] Therefore, it has been suggested that AST levels in GCF may represent a potential marker for monitoring the periodontal metabolism.[1011] These enzymes are reported to be good indicators of bone metabolism and periodontal tissue remodeling associated with orthodontic tooth movement.

In osseous clefts, the osseous structures are absent or poorly developed in the region of the periodontal supportive tissue.[12] Studies have found that clinical attachment levels were similar at alveolar cleft and control sites, i.e., sites not adjacent to the cleft.[13] However, significantly more radiographic alveolar bone loss was observed at cleft sites when compared with control sites. This, in turn, demonstrated the presence of a periodontal attachment apparatus characterized by the presence of a long supracrestal connective tissue attachment. Salvi et al.[14] demonstrated that subjects with orofacial clefts were at high risk for periodontal disease progression. Furthermore, alveolar clefts sites underwent more periodontal tissue destruction compared with control sites over a 14-year period.

Results from past animal studies done in dentitions with reduced periodontium showed that in the presence of plaque; orthodontic forces can cause angular defects and with tipping and intruding movements attachment loss can occur.[15161718] The poorly developed osseous structures in cleft palate patients may be traumatized in the course of orthodontic therapy;[19] hence, require constant monitoring during orthodontic treatment. This study was undertaken to evaluate the tissue response of cleft palate patients during orthodontic treatment and to assess any difference in the tissue response with that of non-cleft patients undergoing orthodontic treatment.

MATERIALS AND METHODS

Study population

A total of 20 patients were recruited from the department of orthodontics and dentofacial orthopaedics A.B. Shetty Memorial Institute of Dental Sciences mean age 19.75 + 2.93. The patients consisted of randomly selected 10 cleft palate patients (group I-3 females and 7 males) and 10 non-cleft patients (group II-5 females and 5 males) who required orthodontic treatment.

Patients with good general health and periodontally healthy individuals with gingival index (GI) score <1 and probing sulcus depth <3 mm were included in the study.

Conditions leading to exclusion from the study were as follows:

Patients who do not follow the given oral hygiene instructions

Prior history of having undergone orthodontic treatment

Gingiva showing signs of inflammation with GI scores >1

Pregnant women and women on oral contraceptives

Patients who have been on any long term medications 6 months preceding the study.

Patients who satisfied the inclusion criteria of the study were selected. Furthermore, each patient received a detailed explanation regarding the study procedure and written informed consent was obtained from those who agreed to participate voluntarily in the study. Ethical approval was obtained from the institutional review board. Each patient underwent thorough oral prophylaxis 1 week prior to the collection of samples. Patients were instructed and supervised for maintaining strict oral hygiene throughout the study The GI was used for the purpose of assessing the severity of gingivitis. The orthodontic treatment was done using fixed appliances with superelastic nickel-titanium wire that gave light continuous forces of approximately 150 cN.

GCF collection

In group I, the incisor adjacent to cleft and molar of the same side were selected as sampling site. In group II, central incisor and molar of the same side were selected as the sampling site.

GCF was collected using volumetric micropipettes (Sigma chemical company St. Louis USA). Before the sample was collected the patients were asked to rinse vigorously with a glass of cold sterile water to cleanse the oral cavity. The isolation of the teeth was obtained using a cheek retractor, suction and cotton rolls. The micro capillary pipette was placed extracrevicularly for 5 min and 1 μl GCF was collected. In case of inadequate sample, collection was repeated.

The samples contaminated with blood, saliva and sites which showed the presence of plaque and inflammation were excluded from the study. GCF was collected at the following intervals - pre-treatment (baseline) 3rd, 9th, 15th and 30th day from the start of orthodontic force application.

Sample preparation

1 μl of GCF was diluted to 100 μl with Sorensens media containing 0.05% bovine serum albumin in phosphate-buffered saline (pH 7.0). The samples were centrifuged at 2000 rpm in a microcentrifuge for 1 min to remove the bacterial and cellular debris. The samples were analyzed immediately or stored at −70°C. The enzyme kits from Raichem for acid phosphatase (ACP), Biocon for AST and alkaline phosphatase were used for quantitative estimation of enzyme activity. This kit is based on the reference method of the International Federation of Clinical Chemistry.

The acid and alkaline phosphatase activities depend upon the hydrolysis of p-nitrophenyl phosphate by the enzymes, yielding p-nitrophenol and inorganic phosphate. When made alkaline, p-nitrophenol is converted to a yellow complex readily measured at 400-420 nm. The intensity of color formed is proportional to phosphatase activity. AST activity is measured photometrically by measuring transamination of aspartic acid and oxaloacetic acid. The enzyme activity was measured photometrically using semi-automated autoanalyser capable of measuring absorbance at 405 nm.

Statistical analysis

The values were calculated as mean ± standard deviation. The difference in mean levels of enzyme activity of the ACP, ALP and AST in GCF samples (U/μl) for both groups was compared using Mann Whitney U test. Students unpaired t-test was used for each enzyme activity for each time interval.

RESULTS

Quantitative analysis of enzyme levels of acid phosphatase, alkaline phosphatase and AST in GCF of cleft and non-cleft palate patients was estimated (U/μl) and compared during orthodontic treatment. In group I, ACP enzyme showed significant (P < 0.001) activity which was 6.1 and 5 fold at incisor and molar sites respectively from pre-treatment till the 3rd day and then showed a decrease in activity towards the 30th day. However, ALP enzyme showed significant (P < 0.001) increase in activity which was 3.5 and 5 fold at incisor and molar sites respectively. The peak in enzyme activity occurred on the 15th day and then showed a decrease in activity towards the 30th day. Similarly, AST enzyme showed 5 and 6 fold gradual increase in enzyme activity from pre-treatment till the 15th day at incisor and molar sites respectively and then showed a decrease in activity towards the 30th day [Table 1 and Figure 1].

Table 1

Mean acid phosphatase, alkaline phosphatase, aspartate aminotransferase enzyme activity in group I patients (mean±SD) (U/μl)

Figure 1

Acid phosphatase, alkaline phosphatase, aspartate aminotransferase enzyme levels in gingival crevicular fluid of group I at incisor and molar sites during orthodontic treatment

In group II ACP enzyme showed significant (P < 0.001) activity, which was 5 and 6 fold at incisor and molar sites respectively. The peak in enzyme activity occurred on the 3rd day and then showed a decrease towards the 30th day. ALP enzyme showed 3 and 5 fold significant activity with a peak in enzyme activity occurring on the 15th day at incisor and molar sites respectively. AST enzyme levels showed 4 fold increase in enzyme activity from pre-treatment till the 15th day at both sites and then showed a decrease towards the 30th day [Table 2 and Figure 2].

Table 2

Mean acid phosphatase, alkaline phosphatase, aspartate aminotransferase enzyme activity in group II patients (mean±SD) (U/μl)

Figure 2

Acid phosphatase, alkaline phosphatase, aspartate aminotransferase enzyme levels in gingival crevicular fluid of group II at incisor and molar sites during orthodontic treatment

Comparison of enzyme activity between sites

Comparison of ACP, ALP, AST enzyme activity between the incisor and molar sites in group I (P = 0.006, P = 0.007 and P = 0.001 respectively) and group II (P = 0.004, P = 0.004and P = 0.004 respectively) patients by Mann Whitney U test showed statistical significant difference in enzyme activity between the sites [Tables 3 and 4].

Table 3

Comparison of enzymatic activity between the incisor and molar sites from base line to 30th day in group I

Table 4

Comparison of enzymatic levels between the incisor and molar sites from base line to 30th day in group II

Comparison of enzyme activity between the two groups

However, comparison between group I and group II for enzyme activity with respect to the particular site i.e., for incisor ACP (P = 0.386), ALP (P = 0.385) AST (P = 0.083) and molar ACP (P = 0.515) ALP (P = 0.065) and AST (P = 0.828) respectively showed no statistical significant difference in enzyme activity between the groups [Table 5].

Table 5

Comparison of acid phosphatase, alkaline phosphatase and aspartate aminotransferase enzyme activity at incisor and molar sites between the groups from base line to 30th day

DISCUSSION

The main reasons for treating patients with CLAP are function and esthetics. This entails long-term tooth preservation in the case of a primarily poor hygiene situation with a predisposition to plaque retention.[13] In case of multiple tooth malpositions, transverse space deficit and a primary cross bite situation; periodontal trauma increases and is detrimental to periodontal health, orthodontic treatment adds to the trauma. The mechanism type and extent of iatrogenic damage through orthodontic therapy has been examined extensively. Positive correlation has been described between pressure exertion and damage to the periodontium caused by orthodontic appliances.[16]

Recent advances in the field of orthodontics have led to evaluation of the contents of gingival cervical fluid as a promising non-invasive method of determining tissue changes in the periodontium.[3] The biochemical analysis of GCF promises as an effective means for monitoring and early detection of periodontal disease.[11] This fluid is an osmotically mediated inflammatory exudate that is found in the gingival sulcus. Orthodontic forces induce the movement of PDL fluids and with them any cellular biochemical products produced from prior mechanical perturbation. Therefore, compression of PDL should cause cellular biochemical by products to appear in the sulcus.[20] Few authors have reported that modulators such as Prostaglandin E2, interleukin-1, tumor necrosis factor α, alkaline phosphatase secreted in GCF are elevated during orthodontic tooth movement.[212223] Therefore, it is speculated that the elevation of cytokines and enzymes in GCF reflects these comprehensive biological responses induced by mechanical stress.

The duration of the study was of 30 days and the time interval for GCF collection was so programmed so as to identify and understand the enzymatic changes during the early stages of orthodontic tooth movement and to coincide with initial and lag phase of tooth movement.[24] In both the groups, GCF showed an early increase in enzyme activity on application of orthodontic force, followed by a decrease enzyme activity coinciding with phases of tissue reaction.

By using Students, unpaired t-test comparison of enzyme activity for the each time interval was done and was found to be statistically significant (P < 0.001).

The ACP enzyme showed peak activity by 3rd day followed by a reversal, this is because the initial cell to be stimulated upon orthodontic force application are osteoclasts which release acid phosphatase upon bone resorption.[6] The levels of ALP enzyme showed a peak by the 15th day and then decreased. The late peaking of ALP could be explained due to delayed osteoblastic activity leading to bone deposition. The fall in activity is related to removal of the hylanized zone. Yokoya et al.[25] reported that osteoclasts on the pressure side increased in number up to the 7th day but fell rapidly by the 14th day. When the enzyme activity was high the tooth movement rate was greater. This implies that the alkaline phosphatase activity followed the rate of tooth movement during the initial phases; this was similar to the results of the study by Insoft et al.[23] and Keeling et al.[6]

AST activity in GCF has been correlated with clinical parameters of periodontal health, including attachment loss, alveolar bone levels and GI. Moreover, it has been demonstrated that an increase in the AST activity in GCF is related to periodontitis activity.[10] According to the data from various studies, it seems to be a little bit difficult to address a reference value of AST in GCF, in our study the increase in levels of the enzyme was considered with reference to the baseline readings. For statistical purposes in order to detect where the apex of the AST levels was, we considered only the differences of values between each time point and the baseline point. The values of AST activity in GCF has been shown to increase significantly from base line to 15 days. This increase of AST activity may be explained as a consequence of tissue remodeling. The compression of the PDL induces hyalinization of the most compressed area. This hyalinised zone is described as an area of focal aseptic necrosis that is resistant to degradation and the level of AST decrease as macrophages start resolving the hyalinised tissues.[26] Although the volume of GCF was not measured, we cannot rule out the possibility that the increased levels of enzymes were, at least in part, associated with an increased GCF volume. Samuels et al.[27] reported that although no gingival health changes occur, the GCF volume can increase significantly during orthodontic treatment.

By using Mann Whitney U test ACP, ALP, AST enzyme activity was compared between the incisor adjacent to cleft and molar sites in group I and group II patients and was found to be statistically significant. Both the sites showed tissue response, with more enzyme activity in the incisor site compared to molar site reason being incisor representing the action site and molar being the anchorage unit representing the reaction site. Inter-comparison of enzyme activity between the group I and group II at incisor and molar was found to be statistically not significant.

ACP, ALP and AST enzymes were expressed in both the groups, during initial tooth movement, in sufficient amounts to be detected in the GCF. The levels of ACP, ALP and AST were affected by the orthodontic forces that cause bone and periodontal tissue remodelling, The increased levels of these enzymes did not seem to be associated with gingival inflammation as no changes were observed clinically during the experimental trial, but seem to reflect the biologic activity that takes place during orthodontic movement. No statistical significant difference was observed in the enzyme activity between the cleft patients compared to controls suggesting that there exists no difference in tissue response between the two groups and the periodontium of the teeth in and around the clefts in cleft palate patients can cope relatively well with long term orthodontic treatment. The study has limitations in terms of sample size and duration. The study is limited to a sample of 20 patients and the duration is of 30 days. The findings of this study need further corroboration on a larger sample size and for a longer duration.

However, the presence of a long supracrestal connective tissue attachment and poorly developed osseous structures mandates constant monitoring and controlled force application during orthodontic treatment. The enzyme analysis of GCF could be an important predictor in orthodontic tooth movement and the calorimetric technique can be reliably employed for estimating the enzyme activity in the GCF during orthodontic tooth movement in patients free from periodontal disease.