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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 11  |  Issue : 6  |  Page : 301-304  

Gingival crevicular fluid levels of RANKL and OPG after placement of collagen membrane with simvastatin in the treatment of intrabony defects in chronic periodontitis


1 Department of Periodontics JKK Nattraja Dental College and Hospital, Komarapalayam, Tamil Nadu, India
2 Department of Oral Surgery, JKK Nattraja Dental College and Hospital, Komarapalayam, Tamil Nadu, India
3 Department of Endocrinology, Post Graduate Institute of Basic Medical Sciences (PGIBMS), University of Madras, Chennai, Tamil Nadu, India

Date of Web Publication28-May-2019

Correspondence Address:
Dr. Thangakumaran Suthanthiran
Department of Periodontics, JKK Nattraja Dental College and Hospital, Komarapalayam 638183, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/JPBS.JPBS_18_19

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   Abstract 

Purpose: The aim of this study was to estimate the Receptor activator of nuclear factor kappa-B ligand (RANKL) and Osteoprotegrin (OPG) levels in gingival crevicular fluid (GCF) after placement of collagen membrane with simvastatin in intrabony defects. Materials and Methods: Sixty subjects were grouped according to the treatment plan as Group I and Group II. Group I included patients with intrabony defects treated with collagen membrane. Group II included patients with intrabony defects treated with simvastatin of 1.5mg concentration incorporated into the collagen membrane. A split-mouth design was planned, in which two contralateral sites with >5mm probing pocket depth and radiographic evidence of bone loss at baseline were chosen. Probing pocket depth was standardized with acrylic stent in all the selected areas. GCF samples were collected at baseline and 21 days. The amount of RANKL and OPG in the samples was determined by commercial ELISA kits (Biomedica Medizinprodukte, Austria). Results: When comparing both the groups, Group II had more statistically significant (P < 0.001**) decrease in the levels of RANKL than Group I. In contrast to RANKL, the OPG levels were significantly increased in patients (Group II) having intrabony defects treated with collagen membrane along with simvastatin. Conclusion: Simvastatin-loaded collagen membrane expressed increased OPG and decreased RANKL levels, which could have a potential role in periodontal regeneration.

Keywords: Gingival crevicular fluid, intrabony defects, OPG, RANKL, simvastatin


How to cite this article:
Suthanthiran T, Annamalai S, Chellapandi S, Puthenveetil S, Dhasthaheer S, Narasimhan S. Gingival crevicular fluid levels of RANKL and OPG after placement of collagen membrane with simvastatin in the treatment of intrabony defects in chronic periodontitis. J Pharm Bioall Sci 2019;11, Suppl S2:301-4

How to cite this URL:
Suthanthiran T, Annamalai S, Chellapandi S, Puthenveetil S, Dhasthaheer S, Narasimhan S. Gingival crevicular fluid levels of RANKL and OPG after placement of collagen membrane with simvastatin in the treatment of intrabony defects in chronic periodontitis. J Pharm Bioall Sci [serial online] 2019 [cited 2019 Jun 18];11, Suppl S2:301-4. Available from: http://www.jpbsonline.org/text.asp?2019/11/6/301/258813




   Introduction Top


Periodontitis is an immunoinflammatory disease of supporting tissues of the teeth, caused by specific microorganisms, resulting in the progressive destruction of periodontal ligament and alveolar bone with pocket formation.[1] Regeneration of lost attachment apparatus is the ultimate goal of any periodontal therapy. Although various bone grafts have been used in the past two decades, it resulted in the formation of long junctional epithelium.

To achieve greater predictability with regenerative therapy requires the introduction of a pharmacological agent that not only hampers tissue destruction but also enhances the regenerative capabilities of periodontal tissues. There was a daunting need for an agent that could provide with the dual benefit of inhibiting bone resorption and stimulating the bone formation, thus leading to the regeneration of periodontium. Then, “statins” came into the field of periodontics. Statins, such as the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoAR) inhibitors, are well-established cholesterol-lowering drugs.[2] They modify the inflammatory cascades through pleiotropic actions at multiple levels. Their pleiotropic actions include anti-inflammatory and antioxidant actions, immunomodulation, and increased angiogenesis and bone formation. Simvastatin administered in the prodrug form, which is much more lipophilic than other statins, can effectively cross cellular membrane barriers by passive diffusion. The low cost and impressive long-term safety profile of this compound makes it a suitable agent in periodontal therapy.[3]

The use of simvastatin to promote new bone formation in periodontitis patients depends on its concentration and appropriate delivery system. There are various forms available of which collagen is biocompatible, is bioresorbable, and adapts easily to the shape of defects. The ability of collagen membrane to stimulate adhesion, chemotaxis, and physiological degradation of progenitor cells, together with the possibility of its own degradation, makes it an ideal material for building the membrane.[4]

Receptor activator of nuclear factor kappa-B ligand (RANKL) interacts with its corresponding receptor RANK on mononucleated osteoclast precursors and induces their activation to multinuclear bone resorbing osteoclasts.[5] The effects of RANKL are blocked by its soluble decoy receptor osteoprotegerin (OPG), thus inhibiting osteoclast differentiation, activation, and survival.[6]

Gingival crevicular fluid (GCF) is regarded as a window for noninvasive analysis of periodontal conditions, including markers of connective tissue and bone destruction. Recent clinical studies have confirmed that both RANKL and OPG can be detected in human GCF, and indicate that RANKL is elevated, whereas OPG is decreased in periodontitis.[7],[8]

To date, there is no information regarding the RANKL and OPG levels after placement of collagen membrane with simvastatin in intrabony defects. Thus, the aim of this study was to investigate RANKL and OPG levels in GCF after placement of collagen membrane with simvastatin in intrabony defects of chronic periodontitis.


   Materials and Methods Top


Study population and clinical examination

A total of 60 subjects were included in this study. Subjects were selected from the outpatients in Department of Periodontics, JKK Nattraja Dental College and Hospitals, Komarapalayam, Tamil Nadu, India. The protocol was reviewed and approved by institutional ethical board. The study-related procedures were explained to the patients before they sign an informed consent form. A split-mouth design was planned, in which two contralateral sites with >5mm probing pocket depth and radiographic evidence of bone loss at baseline were chosen. Probing pocket depth was standardized with acrylic stent in all the selected areas.

Criteria for inclusion were individuals with age limit of 20–50 years of both gender; probing depth of >5mm as assessed by William’s graduated probe, and patients with minimum of two contralateral intrabony defects. Criteria for exclusion were individuals with known systemic diseases, short- and long-term therapies, previous periodontal therapy, known drug allergy, teeth with traumatic occlusion, smokers, pregnancy, and lactating women. Subjects were classified according to the treatment plan as Group I and Group II. Group I included patients with intrabony defects treated with collagen membrane. Group II included patients with intrabony defects treated with simvastatin of 1.5mg concentration incorporated into the collagen membrane.

Collection of Gingival crevicular fluid

The sites were isolated by cotton rolls and gently air-dried to remove saliva. Any supragingival plaque was removed from the sites before GCF collection with a sterile curette. A 1–5 μL sterile glass microcapillary pipette was placed at the opening of the periodontal pocket and left for 30s to draw 2 μL GCF into the microcapillary tube. The sample was discarded if blood was detected within the microcapillary tube. Each GCF sample was immediately placed into a sterile, labeled Eppendorf tube and placed on ice, and then transported to the laboratory for processing. GCF samples were collected at baseline and 21 days. All the samples were immediately stored at 80°C. One examiner made all clinical measurements and collected all microbial and GCF samples.

Analysis of RANKL and OPG in GCF

The amount of RANKL and OPG in the samples was determined by commercial ELISA kits (Biomedica Medizinprodukte). Briefly, 100 μL standard and GCF samples were added to the wells in duplicate; 100 μL detection antibody was added to all wells, except negative control, mixed gently, strips covered with plastic film, and incubated for 16–24h at 4°C. Plates were incubated with 200 μL conjugate for 50min at room temperature (18°C–26°C). The plates were then washed five times and 200 μL of substrate was added and incubated for 30min at room temperature (18°C–26°C) in the dark. The reaction was stopped by the addition of 50 μL stop solution and color was measured in an automated microplate spectrophotometer.

Statistical analysis

The data were compiled in a Microsoft excel sheet and transferred to SPSS software, version 20. Descriptive statistics and inferential statistics were applied. The results obtained were analyzed statistically and comparisons were made within each group using Student’s paired distribution “t-test.” In all tests, P > 0.05 was considered nonsignificant (NS), P < 0.05 as significant, and P < 0.001 as highly significant.


   Results Top


Analysis of RANKL and OPG levels in GCF

RANKL levels were significantly elevated in patients having intrabony defects treated with collagen membrane [123.7±0.24 (pg/mL)] when compared with patients having intrabony defects treated with collagen membrane along with simvastatin [58.0±0.21 (pg/mL)]. When comparing both the groups, Group II had more statistically significant (P < 0.001**) decrease in the levels of RANKL than Group I, as shown in [Table 1] and [Graph 1].
Table 1: Comparison between Group I and Group II based on RANKL (pg/mL) and OPG (pg/mL) using Student’s paired t-test

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,
Graph 1: Comparison between Group I and Group II based on RANKL (pg/mL) and OPG (pg/mL).

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In contrast to RANKL, the OPG levels were significantly decreased in patients having intrabony defects treated with collagen membrane [60.0±0.64 (pg/mL)] when compared with patients having intrabony defects treated with collagen membrane along with simvastatin [120.20±0.61 (pg/mL)]. When comparing both the groups, Group II had more statistically significant (P < 0.001**) increase in the levels of OPG than Group I, as shown in [Table 1] and [Graph 1].


   Discussion Top


The regulation of bone resorption is orchestrated by RANKL and its cognate inhibitor, OPG. Disruption of their balanced expression leads to bone loss, as demonstrated in experimental models of bone destructive diseases, including periodontitis.[9] In our present study, we analyzed the levels of RANKL and OPG in GCF after placement of collagen membrane with simvastatin, in intrabony defects. Simvastatin-loaded collagen membrane expressed increased OPG levels and decreased RANKL levels, which could have a potential role in periodontal regeneration.

This was in accordance with the study done by Han et al.[10] in which they demonstrated that simvastatin inhibited the activity of osteoclasts and stimulated new bone formation in rat periodontal tissues, which resulted in increase in OPG and a decrease in RANKL levels. In support with our study Ayukawa et al.[11] also observed that when artificial bone defects treated with simvastatin showed decreased expression of RANKL levels with fewer osteoclasts.

Pagkalos et al.[12] highlighted that only lipophilic statins (e.g., simvastatin) have these effects on bone, because their lipophilia allows them to cross the cell membrane by passive diffusion mechanisms. They also deliver benefits at the bone level through their anti-inflammatory action and through their angiogenic effect because they are able to stimulate the vascular endothelial growth factor. Ruan et al.[13] reported that simvastatin reduced the expression of iNOS and increased BMP-2 and OPG levels in the periodontal tissue, which affects the bone resorption through the modulation of BMP-2 in osteoblasts. Furthermore, Mundy et al.[14] demonstrated increased BMP-2 expression following treatment of murine (2T3) and human (MG63) cell lines with simvastatin. Collectively, all these findings strengthen our result that simvastatin is associated with increasing OPG concentrations and decreasing RANKL, which could have a protective effect against bone breakdown and periodontal attachment loss.

In conclusion, statins have a broad therapeutic effect beyond that of cardioprotection and potentially show great promise in regenerative therapies. Statins are able to achieve the goal of periodontal regeneration.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Tatakis DN, Kumar PS. Etiology and pathogenesis of periodontal diseases. Dent Clin North Am 2005;49:491-516, v.  Back to cited text no. 1
    
2.
Teng YT, Nguyen H, Gao X, Kong YY, Gorczynski RM, Singh B, et al. Functional human T-cell immunity and osteoprotegerin ligand control alveolar bone destruction in periodontal infection. J Clin Invest 2000;106:R59-67.  Back to cited text no. 2
    
3.
Simonet WS, Lacey DL, Dunstan CR, Kelley M, Chang MS, Lüthy R, et al. Osteoprotegerin: A novel secreted protein involved in the regulation of bone density. Cell 1997;89:309-19.  Back to cited text no. 3
    
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Kataria P, Kaur J, Parvez E. Statins: The paradigm shift in periodontal regeneration. SRM J Res Dent Sci 2014;5:26-30.  Back to cited text no. 4
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5.
Dalcico R, de Menezes AM, Deocleciano OB, Oriá RB, Vale ML, Ribeiro RA, et al. Protective mechanisms of simvastatin in experimental periodontal disease. J Periodontol 2013;84:1145-57.  Back to cited text no. 5
    
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Lacey DL, Timms E, Tan HL, Kelley MJ, Dunstan CR, Burgess T, et al. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 1998;93:165-76.  Back to cited text no. 6
    
7.
Lerner UH. Inflammation-induced bone remodeling in periodontal disease and the influence of post-menopausal osteoporosis. J Dent Res 2006;85:596-607.  Back to cited text no. 7
    
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Mogi M, Otogoto J, Ota N, Togari A. Differential expression of RANKL and osteoprotegerin in gingival crevicular fluid of patients with periodontitis. J Dent Res 2004;83:166-9.  Back to cited text no. 8
    
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Crotti T, Smith MD, Hirsch R, Soukoulis S, Weedon H, Capone M, et al. Receptor activator NF kappab ligand (RANKL) and osteoprotegerin (OPG) protein expression in periodontitis. J Periodontal Res 2003;38:380-7.  Back to cited text no. 9
    
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Han GH, Chen YP, Hou JH, Liu C, Chen C, Zhuang JL, et al. Effects of simvastatin on relapse and remodeling of periodontal tissues after tooth movement in rats. Am J Orthod Dentofacial Orthop 2010;138:550.e1-7.  Back to cited text no. 10
    
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Ayukawa Y, Yasukawa E, Moriyama Y, Ogino Y, Wada H, Atsuta I, et al. Local application of statin promotes bone repair through the suppression of osteoclasts and the enhancement of osteoblasts at bone-healing sites in rats. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009;107:336-42.  Back to cited text no. 11
    
12.
Pagkalos J, Cha JM, Kang Y, Heliotis M, Tsiridis E, Mantalaris A. Simvastatin induces osteogenic differentiation of murine embryonic stem cells. J Bone Miner Res 2010;25:2470-8.  Back to cited text no. 12
    
13.
Ruan F, Zheng Q, Wang J. Mechanisms of bone anabolism regulated by statins. Biosci Rep 2012;32:511-9.  Back to cited text no. 13
    
14.
Mundy G, Garrett R, Harris S, Chan J, Chen D, Rossini G, et al. Stimulation of bone formation in vitro and in rodents by statins. Science 1999;286:1946-9.  Back to cited text no. 14
    


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