Metformin gel in the treatment of generalized aggressive periodontitis

Sohair M Nour; Hoda M Elguindy; Morad A Morad

doi:10.4103/tdj.tdj_59_22

ORIGINAL ARTICLE

Year : 2023 | Volume : 20 | Issue : 2 | Page : 95-104

Metformin gel in the treatment of generalized aggressive periodontitis

Sohair M Nour BDs, MSc, PhD ¹, Hoda M Elguindy², Morad A Morad³
¹ Department of Oral Medicine, Periodontology, Oral Radiology and Diagnosis, Tanta University, Tanta, Egypt
² Department of Oral Radiology and Diagnosis, Faculty of Dentistry, Tanta University, Tanta, Egypt
³ Department of Clinical Pathology, Faculty of Medicine, Tanta University, Tanta, Egypt

Date of Submission	18-Nov-2022
Date of Decision	20-Feb-2023
Date of Acceptance	24-Feb-2023
Date of Web Publication	11-May-2023

Correspondence Address:
Sohair M Nour
Department of Oral Medicine and Periodontology, Tanta University, El Geish Street, Tanta
Egypt

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/tdj.tdj_59_22

Abstract

Objectives
The aim of this study was to assess the adjunctive regenerative effect of metformin (MF) gel in the treatment of stage II grade C periodontitis utilizing β-TCP bone graft in the control group, and β-TCP bone graft mixed with 1% MF gel in the test group on clinical, radiographic, and biochemical parameters.
Patients and methods
Thirty intrabony defects were selected in seven patients who met the inclusion criteria. Patients were randomly distributed in the two study groups and treated with β-TCP bone graft plus collagen membrane in the control group and β-TCP bone graft mixed with 1% MF gel plus collagen membrane in the test group. Clinically, evaluation was carried out by clinical parameters (bleeding on probing, pocket depth, and clinical attachment loss) at 3, 6, and 9 months posttreatment. Biochemically, gingival crevicular fluid concentration of receptor activator of nuclear factor-κB ligand (RANKL) and Osteoprotegerin (OPG) at baseline and 9 months posttreatment were evaluated and RANKL/OPG ratio was calculated. Radiographically, quantitative digital subtraction radiography was used to evaluate bone gain and bone mineral density at 9 months posttreatment follow up period.
Results
The intragroup comparison in the two studied groups demonstrated statistically significant improvement in all clinical parameters (bleeding on probing, pocket depth, and clinical attachment loss) after 3, 6, and 9 months when they were compared to their corresponding values at baseline, while, the intergroup comparison showed that there is no statistically significant difference regarding all clinical parameters at 3, 6, and 9 months posttreatment except pocket depth which differs statistically significant at 3 months posttreatment. The intragroup comparison of RANKL and OPG was statistically significant between baseline and 9 months posttreatment in the two groups but intergroup comparison revealed that there is no statistically significant difference. Regarding RANKL/OPG ratio, the intragroup analysis revealed that there was a statistically significant change in this ratio from baseline to 9 months posttreatment in the test and control group. Additionally, the intergroup analysis revealed that there was no statistically significant difference in RANKL/OPG ratio between the test and control group at baseline and at 9 month posttreatment. The intragroup quantitative digital subtraction radiography analysis demonstrated statistically significant bone gain and increase in bone mineral density in the two studied groups. Also, the intergroup comparison revealed that no statistically significant increase in bone level in the two groups although increase in bone density was statistically significant.
Conclusion
The present study demonstrated that guided tissue regeneration utilizing 1% MF gel combined with β-TCP bone graft showed better radiographic results, when compared with guided tissue regeneration utilizing β-TCP bone graft alone. The adjunctive regenerative action of MF is promising material for periodontal regeneration.

Keywords: IngeniOs β-TCP synthetic bone graft, metformin, stage II grade C periodontitis

How to cite this article:
Nour SM, Elguindy HM, Morad MA. Metformin gel in the treatment of generalized aggressive periodontitis. Tanta Dent J 2023;20:95-104

How to cite this URL:
Nour SM, Elguindy HM, Morad MA. Metformin gel in the treatment of generalized aggressive periodontitis. Tanta Dent J [serial online] 2023 [cited 2023 May 27];20:95-104. Available from: http://www.tmj.eg.net/text.asp?2023/20/2/95/376640

Introduction

Stage II grade C periodontitis is a disease that has a deep impact on the patients' oral health-related quality-of-life ^[1]. The aim of periodontal therapy is to resolve the infectious disease process, prevent progression of the disease and fundamentally, reconstitute the lost tissues ^[2],[3]. While reports of periodontal regeneration denoted that some traditional surgical approaches did not generally lead to periodontal regeneration other treatment modalities were employed including grafting with biomaterials and application of biological agents ^[4]. Also, another approach to attain periodontal regeneration is the use of alternative materials which can induce bone formation in addition to their medical applications in treatment of other systemic diseases, one of these materials is metformin (MF). Metformin HCl (1,1-dimethylbiguanide HCl), a second generation biguanide has been used widely for treatment of type II diabetes mellitus ^[5]. Studies reported that, MF has been shown to induce osteoblasts differentiation and bone formation ^[6],[7]. It was proved that it has bone-protective effects in patients with diabetes mellitus ^[8] as some studies on local drug delivery of MF has proved the potential of osteoblasts to uptake MF ^[9]. Additionally, MF was found to decrease intracellular reactive oxygen species and apoptosis ^[10]. Therefore it was suggested that MF may exhibit a favorable effect on alveolar bone in periodontal therapy ^[11].

Furthermore, different concentrations of MF (0.5, 1, and 1.5%) have been used locally in the treatment of chronic periodontitis. 1% MF concentration had been proved to be the most effective both clinically and radiographically ^[12] as it potentiated the effect of bone graft material in treatment of intrabony defects.

The role of the receptor activator of nuclear factor-κB ligand (RANKL)- Osteoprotegerin (OPG) system in human periodontitis has been elucidated and confirmed that both RANKL and OPG can be detected in human gingival crevicular fluid (GCF) ^[13]. Most of the studies indicate that RANKL levels are increased, whereas OPG levels are decreased in GCF from periodontitis affected sites ^{[14],[15],[16],[17]}. Also, an increase in the relative RANKL/OPG ratio is considered indicative of the occurrence of periodontitis ^[1].

An experimental study investigated the effects of systemically administered MF on alveolar bone resorption and on the RANKL/OPG ratio in rats subjected to experimental periapical lesions. The results showed that MF inhibits the periapical lesions by lowering the RANKL/OPG ratio, subsequently reducing the number of osteoclasts and bone resorption areas in the day 28 compared with the control group ^[14].

In the present study, it was hypothesized that MF can provide additional regenerative effect when it is mixed with traditional bone graft material. Therefore, the aim of this study was to evaluate this treatment modality on clinical, radiographical, and biochemical parameters in stage II grade C periodontitis patients.

Patients and methods

The participants who met the inclusion were selected from the outpatient clinic of Oral Medicine, Periodontology, Oral Diagnosis and Radiology Department, Faculty of Dentistry, Tanta University according to the clinical and radiographic criteria proposed by the 1999 International World Workshop for Classification of Periodontal Disease and Conditions ^[18]. A total of 30 intrabony defects were selected randomly. A computer-generated random allocation system was used to allocate 30 selected sites to each treatment group. The mean age of the selected patients ranged from 18 to 42 years to be included in this study. All procedures were explained, and consent form was signed before treatment and the research protocol was approved by the Research Ethical Committee (REC), Faculty of Dentistry, Tanta University. Sample size calculation was done before the start of the study with a statistical software program by a statistician (I.K.). This calculation indicated that the study containing 30 intrabony defect would have 80% power to detect 1 mm statistically significant differences considering a mean standard deviation of 1.15 regarding the selected parameters throughout the study intervals.

Inclusion criteria

Inclusion criteria were the following: (a) presence of angular periodontal intrabony defects with a clinical attachment loss (CAL) more than or equal to 5 mm measured from cemento–enamel junction till the deepest probing depth. (b) Periodontal pocket depth (PPD) more than or equal to 5 mm. (c) There is no recession or furcation involvement. (d) Optimal compliance was evidenced by no missed treatment appointments and a positive attitude towards oral hygiene.

Exclusion criteria

Exclusion criteria were the following: (a) presence of systemic disease or active infection (diabetes mellitus, cardiovascular diseases, hepatitis). (b) Utilization of drugs such as antibiotics and anti-inflammatories. (c) Periodontal surgery had been previously carried out in the selected sites. (4) The habit of smoking. (5) Pregnancy and lactation.

Materials

Synthetic bone graft (IngeniOs β-TCP) (Elabscience, USA) and collagen membrane ((Biotech Biogen, Italy).
Synthetic bone graft loaded with 1% MF gel and collagen membrane.
Enzyme-linked immunosorbent assay (ELISA kit) (Elabscience, USA) for analyzing RANKL and OPG in GCF.

Pretreatment evaluation

Clinical assessment

The periodontal status of the selected patients was assessed and the measurements of the following clinical periodontal parameters were recorded:

Bleeding on probing (BOP) ^[19].
PPD ^[20].
CAL ^[20].

Radiographic assessment

Standardized periapical radiographs were taken for every patient at baseline and 9 months postoperatively with the paralleling technique using Rinn XCP device and individually fabricated radiographic stent at the selected sites in order to ensure a reproducible images.

Biochemical assessment

RANKL/OPG levels were evaluated in GCF samples. The selected sites were cleared from supragingival plaque, a sterile paper strip was gently inserted into the periodontal pocket until mild resistance was felt and left in place for 30 s.

Quantification of receptor activator of nuclear factor-κB ligand and Osteoprotegerin concentrations in gingival crevicular fluid

The total amount of RANKL and OPG in the GCF samples was determined by ELISA in accordance with the manufacturer's instructions. RANKL/OPG levels were evaluated in GCF. From all sites, GCF samples were taken at baseline and 9 months after completion of the treatment.

To obtain the mean value of RANKL/OPG ratio at baseline and 9 months postoperatively in the two studied groups, individual mean values of GCF RANKL concentration were divided by their corresponding mean values of GCF OPG concentration at baseline and 9 months postoperatively. Consequently, the mean value of RANKL/OPG ratio at 9 months postoperatively was compared to the mean value of RANKL/OPG ratio at baseline in each study group. Moreover, the mean value of RANKL/OPG ratio at baseline and 9 months postoperatively in the test group were compared to its corresponding mean values of RANKL/OPG ratio at baseline and 9 months postoperatively in the control group [Figure 1].

Figure 1 The eppendorf tube containing GCF sample in 7.2 PH phosphate buffered saline.

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The presurgical phase

All the patients were enrolled in phase I therapy. Full mouth scaling and root planing was carried out for all patients each patient was given careful instructions on proper oral hygiene measures. Reinforcement of oral hygiene instructions by patient education and motivation was done 4 weeks after phase I therapy. The patient motivation and compliance determine their completion of the treatment protocol.

Reinforcement of oral hygiene instructions by patient education and motivation was done 4 weeks after phase I therapy. The patient motivation and compliance determine their completion of the treatment protocol. Clinical evaluation was assessed 4 weeks after phase I therapy, the selected intrabony defects were subjected to the surgical phase.

Patient grouping

One month following phase I therapy, the selected sites were randomly classified into two groups. The sites were treated with one of the following treatment modalities as follow:

Group I: synthetic bone graft + collagen membrane (control group).

Group II: synthetic bone graft loaded with 1% MF gel + collagen membrane (MF group).

Surgical procedure

Prior to surgery, the patients were instructed to rinse with 0.1% chlorhexidine gluconate for 30 s. Proper anesthesia were obtained in the selected site by administration of 2% lidocaine 1: 80 000 epinephrine. Sulcular incisions were made to preserve interdental tissue as possible. Buccal and lingual or palatal intrasulcular incisions were performed, and full thickness mucoperiosteal flaps on the facial and lingual or palatal aspects of each tooth, segment, or area involved were reflected. Incisions were designed to preserve as much of the interproximal tissue as possible to allow complete coverage. Vertical releasing incisions were placed when needed at least one tooth mesially or distally apart from the tooth receiving the graft material. Collagen membrane was patronized according to the site of surgery and stabilized at the cemento–enamel junction in both groups. Closure was accomplished using interrupted nonresorbable suture 5/0 silk sutures and periodontal dressing was applied [Figure 2].

Figure 2 1% MF gel mixed with equal amount of IngeniOs β-TCP bone graft material before its placement in the intrabony defects and collagen membrane rehydrated with saline solution.

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Metformin gel formulation

MF gel was developed in Pharmaceutical Technology Department, Faculty of Pharmacy, Tanta University. According to Pradeep (2016) [Figure 3].

Figure 3 1% metformin gel after complete preparation to be preserved for use.

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Postoperative care

All the patients received written instructions included administration of antibiotic, analgesic anti-inflammatory, and antiseptic mouth wash. Maintenance recall visits were scheduled. Supportive periodontal therapy was performed which includes periodontal evaluation, reinforcement of plaque control and medical history was updated. Periodontal probing was not performed before 3 months following surgery to prevent damage to the immature regenerated tissues. The patients were recalled at the 3^rd month, 6^th months, and 9^th month for recording the clinical parameters. In addition, at 9^th month radiographic assessment and GCF sampling were performed [Figure 4],[Figure 5],[Figure 6],[Figure 7],[Figure 8],[Figure 9],[Figure 10],[Figure 11].

Figure 4 Pre-operative panoramic view representing stage II grade C periodontitis in 18 years old female patient.

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Figure 5 Pre-operative panoramic view representing stage II grade C periodontitis in 18 years old female patient.

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Figure 6 Surgical view during placement of bone graft material and collagen membrane.

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Figure 7 9 months follow-up clinical view showing normal healing and tissue response.

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Figure 8 Pre-operative panoramic view representing stage II grade C periodontitis in 36 years old female patient.

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Figure 9 Surgical view after reflection of full-thickness muco-periostal flap showing intrabony defects.

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Figure 10 Surgical view during placement of bone graft material and collagen membrane.

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Figure 11 Post-surgical follow-up clinical view showing normal healing and tissue response.

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Statistical analysis

The collected data were organized, tabulated, and statistically analyzed using Computer Software Statistical Package for Social Science (SPSS), version 20.

Results

Considering the strict oral hygiene maintenance program, patient compliance was satisfactory. All patients tolerated the drug well without complications or adverse reactions to the drug. All periodontal clinical parameters were recorded at baseline and at 3, 6, and 9 months follow up period [Table 1],[Table 2],[Table 3].

Table 1 Comparison of bleeding on probing positive sites at follow up periods

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Table 2 Mean value comparison of probing pocket depth at baseline, 3, 6, and 9 months postoperative intervals

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Table 3 Mean values of clinical attachment level comparison at baseline, 3, 6, and 9 months postoperative study intervals

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Statistical analysis of radiographic evaluation measurements

The intragroup comparison in the two studied groups demonstrated statistically significant improvement in all clinical parameters (BOP, PD, and CAL) after 3, 6, and 9 months when they were compared to their corresponding values at baseline, while, the intergroup comparison showed that there is no statistically significant difference regarding all clinical parameters at 3, 6, and 9 months posttreatment except PD which differs statistically significant at 3 months posttreatment.

[Table 4] displayed the mean values of bone gain surface area and the change in bone density from baseline time to 9 months postoperative interval in the the two studied groups. It was demonstrated that, the mean value of bone gain after 9 months from baseline was 7.26 mm (±1.64) in the control group, and the mean value of bone gain after 9 months from baseline was 8.66 mm (±0.22) in the MF group. Statistical analysis revealed that, there was no statistically significant difference between the two groups concerning mean value of bone gain (Z = 1.68, P = 0.093).

Table 4 Comparison of mean values of bone gain and bone mineral density between the two groups at 9 months follow up interval

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Regarding the change in bone mineral density, the mean value of bone mineral density after 9 months from baseline was 38.74 (±2.63) dots per inch in the control group and the mean value of bone mineral density after 9 months from baseline was 67.68 dots per inch (±8.28) in the MF group. Intergroup comparison revealed that the change in mean value of bone mineral density at 9 months postoperatively was statistically significant (Z = 1.680, P = 0.093).

Statistical analysis of biochemical evaluation measurements

[Table 5] displays the intragroup comparison between the mean values of RANKL in GCF at baseline and at 9 months postoperatively in the two studied groups and the intergroup comparison of the same values at baseline and 9 month postoperatively.

Table 5 Comparison of mean values of gingival crevicular fluid receptor activator of nuclear factor kappa-? ligand concentrations between the study groups at baseline and 9 months follow-up interval

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Statistical analysis demonstrated that, there was a statistically significant difference between mean values of RANKL at 9 months postoperative study interval when compared to this value at baseline (Z = 3.415, P = 0.001).

Similarly, in the MF group there was a statistically significant decrease in the mean value of RANKL concentration in GCF from baseline time to 9 months postoperatively (Z = 2.641, P = 0.008).

Intergroup comparison between the two studied groups showed that, the differences in mean values of RANKL in GCF concentration were neither statistically significant at the baseline (Z = 0.58, P = 0.56) nor at 9 month postoperative interval (Z = 0.832, P = 0.405).

Statistical analysis of Osteoprotegerin evaluation

Regarding OPG evaluation, mean values comparison of OPG concentration in GCF were displayed in [Table 6].

Table 6 Mean values of gingival crevicular fluid Osteoprotegerin concentration between the study groups at baseline and 9 months follow up interval

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In the two study groups, the intergroup comparison revealed that, the GCF mean values of OPG concentrations were significantly increased at 9 months postoperatively when they were compared to their mean corresponding values at baseline. While the intergroup comparison revealed that, at the baseline time and at 9 month postoperatively there was no statistically significant difference in the mean values of GCF OPG concentration.

Statistical analysis of mean values of receptor activator of nuclear factor-κB ligand/Osteoprotegerin ratio

Interestingly, the intragroup analysis in [Table 7] revealed that this ratio changed significantly from baseline to 9 months posttreatment follow up period in group I and group II. Moreover, the intergroup analysis proved that there were no statistically significant differences in RANKL/OPG ratio between test and control group at baseline time and at 9 month follow-up period.

Table 7 Statistical analysis of receptor activator of nuclear factor-?B ligand/Osteoprotegerin ratio in gingival crevicular fluid from test and control group at baseline and 9 month follow-up period

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Discussion

This study was designed to be randomized controlled study, as randomized controlled trial provides the best results when trying to find out if a certain drug is better than the standard treatment for medical conditions, also because randomized controlled trial measures how effective a treatment or diagnostic test and it provides the most reliable answers ^[21].

In this study, IngeniOs β-TCP Bioactive bone substitute is used, as it was designed to counteract the disadvantages of conventional β-TCP. Conventional β-TCP grafting materials have been documented to resorb too quickly for complete bone replacement producing less bone formation than the volume of β-TCP resorbed ^[22].

IngeniOs β-TCP Bioactive synthetic bone particles are an advanced, silicated β-TCP formulation designed to be resorbed in balance with replacement by naturally regenerating mineralized bone. IngeniOs β-TCP Synthetic bone particles are highly porous and offer the potential for increased bioactivity over β-TCP-only grafting materials. In addition, this particle size was designed to allow easy manipulation and stability in the bone defect especially when it was mixed with the MF gel in this study ^[23],[24].

The rationale of using MF gel is based on its promising results in the previous research studies as it has an osteogenic effect, increased osteoblast differentiation, and decreased osteoclastic activity ^[25].

Regarding the selection of concentration of MF gel in this study, it was reported that local delivery of varying concentrations of MF gel into periodontal pockets stimulated a significant increase in the PPD reduction and it was found that 1% MF gel provides the optimum clinical benefits ^[26]. In the present study MF was prepared in the form of 1% MF gel and was mixed with bone graft material in the test group (group II). This study also was carried out to investigate the regenerative capacity of MF and get the benefits of drug combination.

An occlusal stent was used as a fixed landmark to facilitate sequential postsurgical evaluation, in terms of probing PD and CAL to obtain comparable and reliable measurements ^[2].

In the present study, using collagen membrane with graft materials in both groups is based on its hemostatic property, as it aids clot formation which adheres to the root surface, and facilitates wound maturation thereby enhances regeneration ^[27].

In the present study, intragroup comparison revealed that the number of positive sites for BOP was statistically decreased significantly at all follow-up periods when compared to baseline in the control and test group, while the intergroup comparison showed that there was no statistically significant difference between the numbers of positive sites at any follow-up time.

The significant decrease in the number of bleeding sites is a logical result to regular daily home care, in which the patient maintains oral hygiene with combination between adequate tooth brushing and the use of adjunctive antiplaque agents in a mouthwash.

The intragroup results of this study showed significant decrease in clinical parameters (PD, CAL, BOP) from baseline to 3, 6, and 9 months study periods which came in agreement with one study which studied the treatment of aggressive periodontitis ^[27]. Intergroup comparison revealed that there is no statistically significant difference in mean values of clinical parameters at 3, 6, and 9 months follow-up periods in groups I and II, while there was statistically significant difference in mean values of PD at 3 months follow-up period between groups I and II.

Regarding the statistical analysis of PD and CAL mean values, the improvement through the intergroup comparison resulted from the main events of periodontal wound healing which were completed within two to three weeks of wound closure, followed by tissue maturation and re-modeling as the resolution of inflammation is important for the outcome of periodontal treatment. Also, there was a synergetic effect of MF on bone graft material which facilitates the proliferation, and differentiation of periodontal ligament cells in addition to the role of nonsurgical therapy ^{[28],[29],[30]}.

The nonsignificant difference between the test and control group through the intergroup comparison could be attributed to that the effect of MF does not yield sufficient bone regeneration which may be due to the destructive nature of GAP.

On the other side, the intergroup comparison showed that there was a statistically significant difference in PD at 3 months follow-up period which can be explained by the variations in the baseline measurements of PD between the two groups.

In contradiction to the present results, Rao and colleagues proved that local delivery of MF for 6 months resulted in greater decrease in BOP, PD and more CAL gain with statistically significant bone gain in vertical defect sites treated with SRP plus locally delivered MF versus SRP plus placebo in smokers with GAP ^[28].

Also the present results are inconsistent with Pankaj and colleagues who used 1% MF gel as an adjunct to SRP in treatment of intrabony defects with chronic periodontitis. He found that 1% MF gel stimulated a statistically significant PD reduction, CAL gains and improved bone gain when compared with placebo gel ^[31].

Additionally, these results came inconsistent with the study performed by Rao and colleagues who reported that the local delivery of 1% MF gel into the periodontal pocket in smokers stimulates a statistically significant increase in PD reduction in vertical bone defects ^[28].

The intragroup comparison of mean values of bone gain at 9 months and baseline in groups I and II showed that, there was a statistically significant difference. The intragroup comparison of mean values of BMD at 9 months and baseline in groups I and II showed that, there was a statistically significant difference.

Many clinical studies have demonstrated that guided tissue regeneration (GTR) is a successful treatment modality of periodontal reconstructive surgery and it has become an accepted procedure in most periodontal practices including treatment of intrabony defects.

In concern to combination between collagen membrane and bone grafting material in this study, Needleman et al. ^[32] systematically reviewed the evidence for efficacy of GTR for intrabony defects. In this systematic review, randomized controlled trials of at least 12 months follow-up period comparing GTR with open flap debridement were included in this study. It was reported, GTR was more effective than open flap debridement as it improved CAL, reduced PD, decreased gingival recession and resulted in more gain in hard tissue probing at re-entry surgery ^[33]. Additionally, Gineste et al. ^[34] reported a statistically significant increase in new bone formation and connective tissue attachment in surgically created periodontal defects in dogs treated by type 1 collagen barrier.

The intergroup comparison regarding mean values of bone gain at 9 month follow up period for groups I and II revealed that there is no statistically significant difference between the two study groups while, the intergroup comparison regarding mean values of increase in BMD at 9 months follow up period showed that there was a statistically significant difference.

This radiographic results of MF treated group which showed increase in bone formation and BMD could be attributed to that MF induced bone regeneration by induction of osteoblast differentiation and inhibition of osteoclast activity in addition to increase in collagen type I synthesis and osteocalcin expression which is a bone formation marker.

Recently, Arafat and colleagues also evaluated the effects of topical application of MF in bone defects after surgical extraction of mandibular third molar in 20 patients of the test group. The patients underwent surgical extraction followed by application of MF gel in the surgical site. Radiographic evaluation by cone beam computed tomography was performed immediately after operation and at 6 months postoperatively to evaluate BMD. At 6 months postoperatively, concerning the test group, there was a statistically significant improvement regarding BMD at the surgical sites treated by MF gel. Consequently, bone-formative effects of the MF can be considerable ^[27].

To find a reliable molecular marker of periodontal tissue destruction is considered a major challenge in clinical periodontics however many different biomarkers associated with bone turnover have been assessed in GCF. RANK/OPG system plays a significant role in the production and activation of osteoclasts and in the regulation of bone resorption. In the current study, the intragroup biochemical analysis of GCF RANKL and OPG level resulted in decreased RANKL level, increased OPG level and decreased RANKL/OPG ratio in GCF in both groups at 9 months after periodontal surgery.

The findings of the current study came in agreement with Bostanci et al. ^[35] who evaluated the levels of RANKL, OPG and their relative ratio in GCF of healthy and periodontally diseased patients. RANKL and OPG concentrations in GCF were measured by ELISA. It was demonstrated that, RANKL levels were low in healthy and gingivitis groups, although they were increased in presence of periodontitis. On the contrary, OPG levels were higher in healthy periodontal tissue than periodontitis affected tissues. Also, there were no statistically significant differences in RANKL and OPG levels between chronic periodontitis and GAP patients. There was a statistically significant elevation in RANKL/OPG ratio in presence of periodontitis, as the level of RANKL and OPG in GCF were oppositely regulated in periodontitis, resulting in an enhanced RANKL/OPG ratio.

The current results were also supported by the findings of Ibrahim ^[36] who compared the levels of RANKL and OPG and their ratios in GCF from patients suffering from GAP. The results showed higher concentration levels of RANKL, OPG, and RANKL/OPG ratios in the GAP group compared with the control group. Thus, he concluded that, the GCF total amount of RANKL, OPG have a statistically significant increase in periodontal disease supporting its role in the alveolar bone changes developed in this disease.

In explanation to MF osteogenic effect presented by increased BMD and OPG level in GCF at 9 month follow-up period, a study has been performed on biologic transport of MF in osteoblasts and found that osteoblasts could uptake MF. Moreover, MF was found to induce a statistically significant decrease in intracellular reactive oxygen species and had a direct osteogenic effect on osteoblasts ^[37].

The present results were explained by in vitro study carried out by Mai et al. ^[38] who examined the effect of MF on RANKL and OPG system. They found that MF enhanced osteoblasts differentiation, inhibited osteoclast differentiation and prevented bone loss in ovariectomized rats.

Moreover, it was proved that MF could induce the growth and differentiation of osteoblast like cells and can increase the extracellular matrix mineralization ^[39]. MF also was found to decrease risk of fracture in diabetic patients ^[40]. Additionally, it was demonstrated that MF inhibited bone resorption by reducing the number of osteoclasts and lowering the RANKL/OPG ratio ^[10].

In the present study, the synergetic effect of MF on IngeniOs β-TCP bioactive bone graft induced improvement in BMD. Additionally, RANKL/OPG ratio was decreased after periodontal therapy, which indicated that RANKL/OPG ratio is important in the process of periodontal regeneration.

According to this study findings it could be concluded that GTR utilizing 1% MF gel combined with β-TCP bone graft showed better radiographic results, when compared with GTR utilizing β-TCP bone graft alone. The adjunctive regenerative action of MF is promising material for periodontal regeneration.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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