Gingival crevicular fluid osteocalcin level as a biomarker for periodontitis stage III grade B and C response to periodontal treatment after 6 months follow up of Egyptian population sample- Controlled Clinical trial.

doi:10.21203/rs.3.rs-2461633/v1

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Gingival crevicular fluid osteocalcin level as a biomarker for periodontitis stage III grade B and C response to periodontal treatment after 6 months follow up of Egyptian population sample- Controlled Clinical trial.

https://doi.org/10.21203/rs.3.rs-2461633/v1

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Objectives: Periodontitis is characterized by an increased alveolar bone turnover with dominance of bone resorption over bone formation leading to alveolar bone loss and loss of attachment. Osteocalcin is the major noncollagenous calcium-binding single chain protein of bone and dentin matrix and it is a bone marker whose concentration in circulation has been used as a reflector of bone turnover.The present study was conducted to evaluate osteocalcin levels in healthy and diseased periodontium and to correlate the changes in its levels with the changes in the clinical and radiographic parameters before and after treatment.

Materials & Methods: Double blind clinical randomized trial, 40 patients divided into 3 groups; 15 patients diagnosed as periodontitis stage III grade B, 15 patients diagnosed as periodontitis stage III grade C, 10 healthy cases as control. Clinical parameters namely plaque index (PI), Gingival index (GI), Pocket depth (PD), Clinical attachment loss (CAL), radiographic examination, beside gingival crevicular fluid (GCF) samples collection for Osteocalcin level, tested at 3 time points 1 before and 2 after periodontal treatment, which included phase I therapy, and respective periodontal surgery accompanied by systemic antibiotics protocol.

Results: revealed a positive correlation in the changes of osteocalcin levels in relation to probing depth, clinical attachment level, plaque index and gingival index and a negative correlation with the changes in bone density although these correlations were insignificant.

Conclusions: Osteocalcin in gingival crevicular fluid as marker for diagnosis , prognosis, and follow up of the periodontitis infection activity, As a second conclusion cumulative non surgical, surgical and systemic antibiotic medication have high ability in the control of periodontal infection which could be measured clinically and radiographically.

Clinical relevance: Osteocalcin would be suitable for diagnosis and follow up of the disease, but not suitable for the prognosis of periodontal disease infection.

Bone marker

Osteocalcine

periodontal disease

Periodontitis is a chronic inflammatory condition caused by bacteria that affects the soft tissue and bone that surrounds the teeth(al Bahrawy et al 2011). The lack of a healthy balance between microbial virulence agents and the host inflammatory response appears to be the major cause of periodontal disease, according to current knowledge on the pathophysiology of periodontal disease(Amano, 2010). While the immune system defends the host against microbial dental plaque, it also attacks it. During the host-mediated process in response to the bacterial infection, inflammation and tissue damage were early and ongoing occurrences.(ϖ Kornman K, 1986). This end by apical migration of the junctional epithelium, losing connective tissue connection surrounding the teeth, as well as the creation of periodontal pockets characterized it. Alveolar bone turnover is higher, yet bone resorption may outnumber bone production, resulting in alveolar bone loss and attachment loss(Lee et al., 1999a).

Periodontal disorders have diverse etiological causes and development patterns. We can relate variability in host response to microbial infections to the presence of variables that may influence the host response. The extent, sequel, and control of the reaction, and hence the pace of periodontal tissue destruction, differ between periodontitis grade B and periodontitis grade C, to present two forms of inflammatory periodontal disease (Papapanou et al., 2018).

Assessment of clinical factors and radiography were two traditional periodontal diagnosis approaches. However, none of the detection technologies was specific enough to provide a trustworthy tool for identifying and monitoring periodontal disease-related hard tissue deterioration(Armitage GC., 1993). As a result, such approaches might not always detect active local periodontitis. As a result, biochemical supplementary methods to aid in the clinical diagnosis of periodontitis would be beneficial for early detection, identification, and screening(Castro et al., n.d.).

Bone homeostasis maintained by a paired-step; resorption and production process which shows changes in bone turnover. Markers of bone formation were byproducts of collagen production, matrix proteins, or osteoblastic enzymes reflect osteoblast activity(Civitelli et al., 2009). Osteocalcin is a short (5.4 kDa) calcium-binding protein found in bone matrix, accounting for 10–20% of non-collagenous protein. It contains three residues of gamma-carboxyglutamic acid (Gla), a calcium-binding amino acid allows for certain conformational modifications that allow it to attach to hydroxyapatite and accumulate in the bone matrix(Lee et al., 1999b). This vitamin K and D-dependent protein found in the extracellular mineralized matrix of bone and in the serum of circulating blood. Mature osteoblasts, osteocytes, and odontoblasts produce it(Hauschka et al., 1986).

According to Curtis et al. (Curtis et al., 1989), “markers of disease” can fall into three categories: 1) Active disease indicator, 2) predictor of a future disease, and 3) future disease initiation predictor in healthy areas. In gingival crevicular fluid, many bone-related proteins discovered, markers of bone formation such as osteonectin, osteocalcin, bone phosphoprotein, or markers of bone resorption such as type I collagen telopeptides the breakdown product of type I bone collagen, the main component of the organic bone matrix, and a marker of bone resorption, which reflect osteoclastic activity., which could be useful as a marker of bone resorption in periodontal disease

(Eley & Cox, 1998b) (Embery et al., 2000) (Kido et al., 2001)

Serum osteocalcin thought to be a reliable indicator of bone turnover. It’s the most important non-collagenous calcium-binding single-chain short protein with 49 amino acids that’s high in glutamic acid (GLA) in the bone matrix(Giannobile et al., 2003)(Seibel, 2005)

Enzyme-linked immunosorbent assays (ELISA) developed for a wide range of diagnostic tests involving biomolecule measurement in body fluids. We would test Osteocalcin in plasma or serum using an immunoassay, and different antibodies identify different fragments, and used as a marker for dental practice. For many years, osteocalcin thought to be the only marker that could represent bone development, and it is still employed

(Eley & Cox, 1998a)

Thus the aim of this study was to measure the effect of periodontal treatment, including non surgical conservative periodontal treatment and surgical periodontal treatment resective flap surgery, on the progress of different form of periodontitis; grade B and grade C which differ in the rate of periodontal deterioration. Periodontal clinical parameters and osteocalcin bio marker was the caliber used to measure the effect of intervention in this study. The study population, although was small but represented the Egyptian population with no bias to age or sex.

All subjects took part in this study recruited from the outpatient clinic of oral medicine and periodontology department of Cairo university Egypt. Forty patients with age range between 25 to 45 years, 12 males, 28 females selected. We divided patients into 3 groups depending on the world's workshop classification system 2017(Papapanou et al., 2018).

1. Group 1: 15 patients diagnosed as periodontitis stage III grade B

2. Group 2: 15 patients diagnosed as periodontitis stage III grade C

3. Group 3: 10 healthy cases as control.

We recruited subjects for this study according to the following inclusion criteria:

1. Patients must not have subjected to any chemical or mechanical periodontal therapy for at least 6 months before being involved in the study.

2. Patients must not have treated with any medication that affects the bone turnover.

3. Patients must not show any history of any systemic disease that affects bone turn over

4. Female patients must not be pregnant or lactating or in menopause during participating in the study.

5. Patients must not be a smoker or with a history of smoking.

6. Patients must not have any history of allergic reactions to any medications used in the study.

The study design was parallel randomized clinical trial, where periodontal clinical parameters

recorded at baseline and six months after treatment, for each tooth at 4 points:

Disto-buccal, buccal, Mesio-buccal and lingual surfaces. The periodontal clinical

parameters that recorded were: Plaque index (PI), Gingival index (GI), Pocket

depth (Pd), and clinical attachment loss (CAL).

1. Plaque index (PI)(Papapanou et al., 2018)

(LOE & SILNESS, 1963)

The scores from 0 to 3:

0 = Gingiva was free of plaque.

1= No plaque detected by naked eye, but we could see plaque on probe tip when moving probe on gingival crevice.

2 = A moderate to thick plaque seen by naked eye in the gingival cervical area.

3= Heavy plaque accumulation that cover the gingival crevice.

2. Gingival index (GI)(Papapanou et al., 2018)

(SILNESS & LOE, 1964)

Severity of gingival inflammation scored from 0 to 3:

0= No inflammation detected.

1= Mild change in gingival color and texture.

2= Moderate gingival swelling, redness, edema and bleeding with probing.

3= Marked gingival swelling, redness and spontaneous bleeding.

3. Pocket depth (PD)

Using William’s graduated periodontal probe, the distance between the gingival margin and the deepest point of the pocket measured, where the probe was inserted parallel to the vertical axis of the tooth.

4. Clinical attachment loss (CAL)

Using William’s graduated probe the distance between the cement- enamel

junction and the deepest point of the pocket measured.

Radiographic examination:

The radiographic images standardized using digital periapical imaging using parallel cone technique

(Bean, 1969) (Plotnick et al., 1971) (Mourshed & McKinney, 1972

images taken at baseline and 6 months after surgery, by using intra oral sensor holder standardization of images preserved.

We obtained quantitative values of bone changes in the defect area before and after treatment by the sensor software (Digora system, Soredex, Orion Corp., Finland). The software quantified the values using the optical density of the radiographic image the pixels and gray level which were given numerical data values. We tabulated the mean of this values for further statistical analysis.

During the radiographic exposure, film holders were used to stabilize the distance between the patient and the x-ray source, ensuring the standardization and reproducibility of the exposure parameters, as well as the accuracy of dental radiographs and avoiding beam miss-angulation, which may appear to affect bone measurements

(Rushton & Horner, 1994, 1995)

Gingival crevicular fluid samples:

We got samples of the gingival crevicular fluid in mid-morning hours(Ladlow et al., 2002a), where samples collected from the most affected site of the patient. Another sample taken from healthy sites to be a control. We collected gingival crevicular fluid samples according to Wilson et al. protocol (Wilson et al., 2003a) , where the test sites well isolated and dried tharaly, sterilized filter paper strip inserted at the gingival crevice for 30 seconds, Filter paper strip then inserted in a micro-centrifuge vial and stored at -70 degree Celsius for laboratory investigation.

Periodontal therapy:

After collection of baseline date, all patients subjected to supportive periodontal mechanical treatment supra-and sub gingival scaling (Phase I therapy), beside chemical treatment Augmentin 625 mg (GalaxoSmithklien) and Flagyl (Sanofi aventis) 250 mg 3 times per day for 1 week.(Pavicić et al., 1991, 1994a) Patients motivated and instructed about the oral hygiene measures tooth brushing and interdental cleansing. After 2 months patients recalled evaluating their oral hygiene status. Sites with pocket depth more than or equal to 5 mm subjected to resective open flap debridment periodontal surgery (Nickles et al., 2009)

Surgical intervention:

A primary incision made 1 mm apical to the gingival margin with 45 degree angulation apically (reverse bevel incision), scalloping and festooning of the incision followed to keep the normal gingival architecture. We made secondary releasing incisions at the line angles. Gingiva was reflected using muco-periosteal elevator. We made sulcular incision from the base of the pocket to bone to release the infected gingival wedge that contains the infected periodontal pocket. We made a final tertiary incision coronal to the interdental bone, the gingival collar removed by a curette.

The tooth root carefully scaled, yet buccal and lingual flaps sutured together by interrupted suture, and we adapted periodontal pack to the wound area. Sutures removed after 10 to 14 days, we instructed Oral hygiene measure using interdental tooth brush

(SLOTS & JORGENSEN, 2000)

A follow up routine weekly for 1 month, monthly for 6 months, to ensure that patients’ plaque index and gingival index scores were between 0 to 1 in the surgical sites, where supragingival mechanical plaque removal performed when necessary.

Laboratory investigation.

1. Gingival crevicular fluid samples retrieved from - 70 degree and left to warm, centrifuged at 15000 mg for 5 minutes with aliquots of 200 ul phosphate buffer, this procedure repeated twice.

2. In vitro immune enzymatic kit for quantification of human osteocalcin protein used to measure concentration of osteocalcin in gingival crevicular fluid samples (BioSource hOST-EASIA Kit BioSource Europe S.A.Rue de l’Industrie, 8, B-1400 Nivelles, Belgium)

3. Osteocalcin quantity in the samples measured in pg/ml and plotted in a result curve, data mean and standard deviation measured.

The principle of the method:

The ELISA kit was micro titer plate as a solid phase amplified sensitivity immune assay.

A mono clonal antibody against human osteocalcin was the principle of this assay, monoclonal antibody 1 attached to the micro titer well, and which fix the osteocalcin of the sample, then mono clonal antibody 2 which is labelled with horseradish peroxidase (HRP)

After the incubation period to allow sandwiching between the monoclonal antibodies and the enzyme, we washed the micro titer plate to remove the unbound sample or antibody.

We add a chromogenic solution to measure the HRP bound antibodies to human osteocalcin enzyme in the sample.

We added a stop solution to stop the chromogenic reaction, the plate subjected to a light with a certain wavelength to show the chromogenic solution. The colorimetric absorbance, which was proportional to the substrate concentration, measured the amount of concentration of the human osteocalcin in the sample.

A calibration curve plotted the osteocalcin concentration determined by the interception with the calibration curve.

Statistical analysis:

Data first evaluated for normality using Kolmogorov-Smirnov and Shapiro-Wilk tests, student t test and paired t test, one-way ANOVA used to compare between means of the three groups of parametric data, while Duncan’s test used to compare significance between individual groups. For non-parametric data Mann Whitney U test to compare between means of different groups and Wilcoxon signed-rank test to compare data in change of time of each group. Pearson’s correlation coefficient.

In this study. Mild to moderate inflammation noticed after the surgical phase, that sustained for three to four weeks during the maintenance phase. Getting that, the irritation subsided, and a continued focus on oral cleanliness and follow-up scaling treatments allowed the inflammation to recover. Probing depth, Clinical attachment loss., Bone density, and Osteocalcin level, showed normal distribution values according to Kolmogorov-Smirnov and Shapiro-Wilk tests. Student t-test, paired t-test, and ANOVA test were used to compare the difference of means between groups. Gingival index, plaque index values not normally distributed then Mann-Whitney U test and Wilcoxon signed-rank test used for comparison of means between different groups.

1. Changes in clinical parameters:

I-Changes in Plaque index:

At baseline, the Plaque index in group 1 measured 1.6 ±0.8, and after six months, it was 0.7 ±0.5. The mean plaque index in group 2 measured 1 at baseline and 0.4 ±0.5 at six months. The Mann-Whitney U test revealed a statistically significant difference between the two groups at baseline. Table 1 figure 1 The mean change in plaque index showed 0.9 ±0.7 in group 1 and 0.6 ±0.5 in group 2. After six months, there was a statistically significant difference in mean plaque index between the two groups. Table 2 figure 2. The mean percentage decrease in plaque index in group 1 measured 53.5 percent (±41.4), while in group 2 it was 60 percent (±51.6). Using the Mann-Whitney U test, there was no statistically significant difference in the mean percentage change after six months between the two groups. Table 3 figure 3

II-Changes in the gingival index

At the start of the study, the gingival index in group 1 measured 1.8 ±0.9. After 6 months, it was 0.7 ±0.5. The average GI in group 2 measured 1.3 ±0.5 at baseline and 0.6 ±0.5 after 6 months. Using the Mann-Whitney U test, there was no statistically significant difference between groups at baseline and at 6 months. Table 4 figure 4. In group 1, the mean change in gingival index measured 1.1 ±0.9, while in group 2, it measured 0.7 ±0.5. After six months; the Wilcoxon signed rank test revealed a substantial decrease in the mean gingival index of the two groups. Table 5 figure 5. The mean percentage change in the gingival index was 55 percent ±41.6 percent in group 1 and 55 percent ±43.8 percent in group 2. Using the Mann-Whitney U test, there was no statistically significant difference in the mean percentage change of Gingival index after six months between the two groups. Table 6 figure 6.

III-Changes in probing depth:

The average probing depth in group 1 measured 6.4 ±1.1mm at baseline and 2.9 ±1.3mm after six months. The average pocket depth in group 2 measured 9.2 ±1.3mm at baseline and 4.3 ±1.2mm at six months. Using the student’s t-test, group 2 had a statistically significantly higher mean probing depth than group 1 at baseline and after six months. Table 7 figure 7. The average change in probing depth measured 3.5 ±0.7 mm in group 1 and 4.9 ±0.6 mm in group 2. Using the paired t-test, there was a statistically significant decrease in mean probing depth between the two groups after six months. Table 8 figure 8. The average percentage change in probing depth measured 55.1 percent ±9.1 in group 1 and 54 percent ±8.7 in group 2. Using the student t-test, there was no statistically significant difference between the two groups in the mean percentage change of probing depth after six months. Table 9 figure 9.

IV-Changes in clinical attachment level:

The mean clinical attachment level in group 1 measured 4.8 ±0.8 mm at baseline and 2.2 ±1 mm at six months. The mean clinical attachment level in group 2 measured 8.1 ±1.8 mm at baseline and 3.5 ±1.3 mm after six months. Using the student’s t-test, group 2 had a statistically significantly higher mean clinical attachment level than group 1 at baseline and after six months. Table 10 figure 10.

The mean change in the CAL measured 2.6 ±1.2mm in group 1 and 4.6 ±0.9mm in group 2. Using the paired t-test, there was a statistically significant decrease in the mean clinical attachment level after six months in both groups. Table 11 figure 11. In group 1, the mean percentage change in clinical attachment level was 53.5 percent ±20.7, while in group 2, it measured 57.5 percent ±8.8. After six months, there was no significant difference between the two groups’ mean percent changes in clinical attachment level. Table 12 figure 12.

1. Changes in radiographic parameters:

Group 1 showed a mean bone density of 101.4 ±5% at baseline and 112.9 ±5% six months later, while group 2 showed a mean bone density of 46.1 ±7% at baseline and 70.8 ±10.3% after six months. Corresponding to a student’s t-test comparison of the two groups’ bone densities at baseline and after 6 months, group 1 had significantly higher mean bone density than group 2. Table 13 Figure 13.

Group 1 showed a mean change in bone density of -11.5 ±5.3, while group 2 showed a mean change of -24.7 ±10.7. According to a paired t-test, the mean bone density of the two groups increased considerably after six months. Table 14 figure 14.

The mean percentage change in bone density in group 1 was 11.5 ±5.9, while it was 56.1 ±29.1 in group 2. According to the Student’s t-test, group 2 had a significantly higher mean percentage change in bone density than group 1. Table 15 figure 15

1. Changes in Osteocalcin level:

The mean osteocalcin level in group 3 measured 871.8 ±208.2 pg/ml, in group 1 was 1173 ±454.4 pg/ml at baseline and 692.8 ±212.1 pg/ml six months after treatment, and in group 2 was 816.7 ±284.1 pg/ml at baseline and 450.5 ±265.6 pg/m after six months. The ANOVA test revealed a significant difference between the three groups at the baseline. Group 1 had the greatest mean level, but there was no significant difference between it and group 3, according to Duncan’s test, whereas group 2 had the lowest mean level. Table 16 figure 16.

The mean change in osteocalcin level was 436.5 ±319.6 in group 1 and 332.9 ±247.2 in group 2 (p < 0.05). A paired t-test for changes in mean osteocalcin levels between the two groups at 6 months revealed a significant decrease in mean osteocalcin levels between the two groups. Table 17 fig 17. The mean percentage change in osteocalcin level in group 1 was 38% ±13.3 and 44.7 ±22.8 in group 2, respectively. After six months, statistical analysis revealed no statistically significant difference in mean percentage changes in osteocalcin levels between the two groups. Table 18 figure 18.

5-Correlation between clinical parameters and osteocalcin level:

Although a positive correlation between plaque index, gingival index, pocket depth, clinical attachment level, and osteocalcin level and a negative correlation with bone density, these correlations not statistically significant, according to Pearson’s correlation coefficient. Table 21.

Cementum, functionally orientated periodontal ligament, alveolar bone, and gingiva make up the native periodontium. This anatomical structure may be lost or damaged because of pathologic and/or traumatic insults(al Bahrawy et al., 2020).(Polimeni et al., 2006) Identifying an unambiguous sign of current periodontal disease well established.(Lee et al., 1999b) Several crevicular biochemical markers have reported to be useful in detecting and/or predicting periodontal disease activity(Nakashima et al., 1996) .In both human and animal studies, osteocalcin recommended as a bone marker. Because commercial kits available, osteocalcin is better suited to these investigations(Kunimatsu K et al., 1993).(Nakashima et al., 1994) (Giannobile et al., 1995)

The goal of this study was to look into gingival crevicular fluid osteocalcin levels as a periodontitis marker by detecting levels of osteocalcin in gingival crevicular fluid from healthy and diseased sites in subjects with severe periodontitis grade B and C, evaluating changes in osteocalcin level in relation to changes in probing depth, clinical attachment level, gingival index, plaque index, and bone density before and after treatment in all groups. Because the quantity of bone markers fluctuates during the day, sampling done at the same time every day, in the middle of the morning. (Ladlow et al., 2002b)

The current study’s design included two types of periodontal diseases: severe periodontitis grade B and C. To simplify comparison of the study’s key criterion (osteocalcin), patients in both groups were within the same age range, had similar general health state, and prescribed the same treatment technique. We prescribed adjunctive combination antibiotic medication as directed by previous studies(van Winkelhoff et al., 1996)(Pavicić et al., 1994b) (Walker & Karpinia, 2002)

Although serum, saliva .and gingival crevicular fluid have been used to assess a participant risk of developing periodontal disease and to track the host’s response to periodontal therapy. Saliva osteocalcin level indicator(Shazam et al., 2020) couldn’t show a significance nor correlation with periodontal infection severity, in other study (Joseph et al., 2020)found that 15.25 ng/mL osteocalcin salivary concentration was best discrimination between healthy and smoking periodontitis, 16.45 ng/mL was excellent descriminator when pocket depth at 6 mm was a cut off, while 19.24 ng/mL concentration was the best when at cut off bone loss of 33.33%.

In this study the osteocalcin level measured in gingival crevicular fluid rather than serum because gingival crevicular fluid is more closely approximated to the periodontal tissues where periodontal disease begins and thus reflects the state of bone tissue more accurately(Ducy et al., 1996).(Ducy et al., 2000) (Wilson et al., 2003b)(Ozmeric, 2004)

To limit the potential effect of proteases from both the host and the bacterium, samples kept cold after collection and swiftly moved to -70 C for analysis. Prior to analysis, we extracted the fluid contained in the filter paper. The fluid collected and preserved until it came time to analyse it. (Wilson et al., 2003b) Following the surgical procedure, detailed supportive periodontal therapy and dental hygiene practices applied.

Because we normally recognise hard tissue changes within six months of treatment, a six-month term chosen for the start of the clinical evaluation of the treatment outcome. Lindhe and co workers (Lindhe et al., 1987); had suggested this follow-up period. Yukna, (Yukna, 1990)and Richardson et al(Richardson et al., 1999) studies had suggested this time period to be the standard for this type of research, regardless of whether the therapy delivered was natural materials or surgical debridement. The presurgical average pocket depth for both treatment groups in this study measured > 5mm, showed that total plaque removal by mechanical scaling and root planing would be improbable (Grisi et al., 2006).

Periodontal surgical therapy an important phase of treating patients with periodontal disorders (Wennström J et al., 2003). The rationale for resective flap method, as described by

(Ramfjord & Nissle, 1974), to be used in this investigation as it enhanced access to root surfaces and supporting bone while also allowing the removal of pocket epithelium, which promotes healing and osteocalcin would be an accurate representation of bone healing. According to Christgau et al.(Christgau et al., 2007), a robust remodelling activity could detected along the surfaces and in the superficial marrow spaces of the inter-radicular bone after surgery. After this technique, a long thin junctional epithelium extending apically interposed between the root surface and the gingival connective tissue heals after 21 days, which considered a stable treatment success (Caton et al., 1980) (Listgarten & Rosenberg, 1979)

The current findings showed that, when compared to baseline, both research groups showed statistically and clinically significant improvements in hard and soft tissue parameters. Six months after treatment, statistical analysis of the data revealed no significant differences in clinical parameters between the two groups; We attribute this to the comprehensive treatment modality and oral hygiene maintenance that covered the major etiologic factors of both disease categories.

Significant quantities of osteocalcin found in gingival crevicular fluid in the current study, which agrees with the findings of (Wilson et al., 2003b)Wilson et al. work seeking to establish a method to evaluate various indicators of bone turnover, in which osteocalcin utilized as a marker of bone turnover.

Periodontitis grade B group in the current study had the lowest mean osteocalcin level at baseline, which could be explained by sampling during a quiescent phase of bone degradation of this group, when osteocalcin production was low. Nakashima et al.(Nakashima et al., 1994), found that total osteocalcin amounts from strips soaked with gingival crevicular fluid from periodontitis sites were significantly higher than those found in healthy and gingivitis sites, in agreement with the findings of this study. Bullon et al. (Bullon et al., 2005), assessed serum, plasma, and gingival crevicular fluid levels of osteocalcin and correlated them with periodontitis and osteoporosis, and found that gingival crevicular fluid level of osteocalcin in periodontitis group was significantly higher than healthy group, which was in agreement with the results of the current study .

In contrast, Lee et al.(Lee et al., 1999a) reported no differences in gingival crevicular fluid osteocalcin levels between diseased and healthy sites in the same patients in a cross-sectional study aimed at detecting the levels of osteocalcin in gingival crevicular fluid from healthy and diseased subjects with periodontitis in order to further investigate its potential role as a possible marker of the disease process. In addition, Isik et al.(Isik et al., 2005), searched changes in bone turnover markers such as deoxypyridinoline (Dpd), osteocalcin, n-telopeptide (NTx), and bone alkaline phosphatase (balp) during experimental orthodontic intrusion of maxillary premolar teeth. Only Dpd values showed statistically significant changes over time.

In this study, the levels of osteocalcin in both study groups were dramatically changed after periodontal therapy. The findings of this study were in contrast to those of Golub et al.,(Golub et al., 1997) which found no changes in osteocalcin levels following periodontal therapy in patients with periodontitis. The likely explanation could be linked to the supplementary use of antibiotic therapy in the current trial, which aimed to eliminate microbial insult and create an environment that promotes periodontal healing.

The current study found a positive correlation between change in osteocalcin level and gingival index (GI) from baseline to six months after treatment, which is consistent with a study by Kunimatsu et al(Kunimatsu K et al., 1993). which found higher levels of osteocalcin in subjects with GI = 3 as compared to subjects with GI scores 0–2 in a cross-sectional study of 14 patients with periodontitis and five patients with gingivitis.

Although a positive association between osteocalcin level and gingival index found in the current study was insignificant, contradicting the findings of Nakashima et al.(Nakashima et al., 1994), which found that osteocalcin showed positive correlations with gingival index. A rational explanation for this finding would be various cytokines and growth factors found in inflammatory periodontal tissue, could encourage osteoblasts to produce osteocalcin. It postulated that osteocalcin synthesis and bone turnover might up-regulated early in inflammation, before bone resorption increases (Murata et al., 2002).

After six months, a substantial increase in mean bone density in both groups, which adversely connected with the amount of osteocalcin in the current study. These findings were consistent with those of a pilot study conducted by Reinhardt et al. (Reinhardt et al., 2004) to investigate local biochemical markers of bone turnover and their relationship to subsequent increased density of healing alveolar bone defects, in which osteocalcin levels were higher at baseline and inversely related to increasing bone density of the healing bone defect. The levels of osteocalcin were higher at the beginning, and negatively related them to the healing bone’s increasing density.

The periodontitis grade C group had a larger mean percentage change in bone density than the periodontitis grade B group in this study. It was plausible to suppose that therapy with combination antibiotics resulted in a speedy cessation of bone destruction because of their effect on the aggressive microbial flora and/or inflammatory cytokines responsible for the disease’s aggressive nature, promoting faster bone regeneration. Another explanation could be because of the study’s length of time, as long-term bone density changes were easier to detect.

The current investigation found a positive (insignificant) correlation between changes in osteocalcin level and probing depth, which is consistent with Nakashima et al., (Nakashima et al., 1996), which found a significant correlation between osteocalcin levels and pocket depth. Bullon et al. (Bullon et al., 2005) discovered that the mean probing depth and clinical attachment level strongly connected to the osteocalcin level, and that osteocalcin level in gingival crevicular fluid corresponds well with periodontitis. There was a considerable improvement in clinical attachment level assessments, which was favorably connected with the change in osteocalcin level; however, this contradicted a study by Nakashima et al, (Nakashima et al., 1994), which found the correlation to be negative.

From this study we could conclude that gingival crevicular fluid osteocalcin levels was unable to distinguish between active and inactive areas, which consistant with Nakashima et al.(Nakashima et al., 1996) study. However, when the biochemical markers prostaglandin E2 osteocalcin, 2-macroglobulin, collagenase, elastase, and alkaline phosphatase assessed together, diagnostic sensitivity and specificity enhanced by 80 and 91 percent. Giannobile et al.(Giannobile et al., 2003) found that osteocalcin has a potential role as a bone-specific marker of bone turnover but not as a prognostic sign of periodontal disease.

Gingival crevicular fluid osteocalcin had potential role as a spcecific marker of bone turnover in periodontal disease. Changes in osteocalcin level in gingival crevicular fluid correlated with improvement in the clinical parameters, and hence osteocalcin as a diagnostic marker had high sensitivity, but its specificity wasn’t obvious in this study. The level of osteocalcin in gingival crevicular fluid can be used in monitoring the response to periodontal therapy, hence osteocalcin was not suitable as a prognostic marker for periodontal disease.

I declare that the authors have no competing interests as defined by Springer, or other interests that might be perceived to influence the results and/or discussion reported in this paper.The results/data/figures in this manuscript have not been published elsewhere, nor are they under consideration (from you or one of your Contributing Authors) by another publisher.All of the material is owned by the authors and/or no permissions are required.

Conflict of interest: This study was self funded and no funding, provision of study materials, medical writing, article processing charges, or grant have been given to the authors by any means. There was no financial and non-financial conflict

Ethical approval and informed consent :

The Cairo University ethical committee approved this study, a written informed consent was obtained from the study participants.

Funding:

No funding was obtained for this study

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Table (1): Comparison in plaque index between the two groups

*: Significant at P ≤ 0.05

Table (2): Changes in plaque index after 6 months within each group

Group	Period	Mean difference	SD	P-value
Group 1	Baseline – 6 months	0.9	0.7	0.014*
Group 2	Baseline – 6 months	0.6	0.5	0.014*

*: Significant at P ≤ 0.05

Table (3): Comparison between mean % changes in plaque index

*: Significant at P ≤ 0.05

Table (4): Comparison in gingival index between the two groups

*: Significant at P ≤ 0.05

Table (5): Changes in gingival index after 6 months within each group

Group	Period	Mean difference	SD	P-value
Group 1	Baseline – 6 months	1.1	0.9	0.015*
Group 2	Baseline – 6 months	0.7	0.5	0.008*

*: Significant at P ≤ 0.05

Table (6): Comparison between mean % changes in GI

*: Significant at P ≤ 0.05

Table (7): Comparison in probing depth between the two groups

*: Significant at P ≤ 0.05

Table (8): Changes in probing depth after 6 months within each group

Group	Period	Mean difference	SD	P-value
Group 1	Baseline – 6 months	3.5	0.7	<0.001*
Group 2	Baseline – 6 months	4.9	0.6	<0.001*

*: Significant at P ≤ 0.05

Table (9): Comparison between mean % changes in PD

*: Significant at P ≤ 0.05

Table (10): Comparison in CAL between the two groups

*: Significant at P ≤ 0.05

Table (11): Changes in CAL after 6 months within each group

Group	Period	Mean difference	SD	P-value
Group 1	Baseline – 6 months	2.6	1.2	<0.001*
Group 2	Baseline – 6 months	4.6	0.9	<0.001*

*: Significant at P ≤ 0.05

Table (12): Comparison between mean % changes in CA

*: Significant at P ≤ 0.05

Table (13): Comparison in bone density between the two groups

*: Significant at P ≤ 0.05

Table (14): Changes by time in bone density within each group

Group	Period	Mean difference	SD	P-value
Group 1	Baseline – 6 months	-11.5	5.3	<0.001*
Group 2	Baseline – 6 months	-24.7	10.7	<0.001*

*: Significant at P ≤ 0.05

Table (15): Comparison between mean % changes in bone density

*: Significant at P ≤ 0.05

Table (16): Comparison between Osteocalcin levels in the three groups

*: Significant at P ≤ 0.05, Means with different letters are statistically significantly different according to Duncan’s test results

Table (16): Comparison between Osteocalcin levels in the three groups

*: Significant at P ≤ 0.05, Means with different letters are statistically significantly different according to Duncan’s test results

Table (17): Changes in Osteocalcin level after 6 months in the study groups

Group	Period	Mean difference	SD	P-value
Group 1	Baseline – 6 months	436.5	319.6	0.001*
Group 2	Baseline – 6 months	332.9	247.2	0.001*

*: Significant at P ≤ 0.05

Table (18): Comparison between mean percentage changes in Osteocalcin level

*: Significant at P ≤ 0.05

Correlation between osteocalcin level and clinical parameters

Table (19): Results of Pearson’s correlation coefficient:

No competing interests reported.

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Gingival crevicular fluid osteocalcin level as a biomarker for periodontitis stage III grade B and C response to periodontal treatment after 6 months follow up of Egyptian population sample- Controlled Clinical trial.

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