The Effect of Using Coenzyme Q10 With Non-Surgical Periodontal Treatment on MMP9 Gingival Crevicular Fluid Level in Patients With Periodontitis: Randomized Controlled Trial

Introduction:

Periodontitis is an irreversible inflammatory condition resulting in a loss of connective tissue attachment and bone destruction which ultimately can lead to loss of the involved teeth (Manthena et al., 2015). It was proven that periodontitis is caused by the immune response products stimulated by microbial plaque around the gingival margin (Preshaw et al., 2004).

In the pathogenesis of the periodontal disease, gingival crevicular fluid (GCF) which is an inflammatory exudate significantly increases (Beklen et al., 2006). This is accompanied by the migration of neutrophils and macrophages with the subsequent release of different matrix metalloproteinases (MMPs) from the neutrophils’ granules (Buduneli & Kinane, 2011). MMPs are endogenous enzymes that can degrade extracellular matrix (ECM) components thus playing an important role in various biological and pathological processes (Anshida et al., 2020).

In human beings, the MMP family is formed of 23 members divided into six groups based on the substrate. These are: collagenases, gelatinases, stromelysins, matrilysins, membrane -type MMPs, and others (Visse & Nagase, 2003). MMPs are considered the most significant elements associated with periodontal disease destruction and can be considered a risk factor for periodontal diseases. MMP-9 is a 92 kDa type IV collagenase released from fibroblasts, neutrophils macrophages, and periodontal pathogens (Koromantzos et al., 2012).

Elevated MMP-8 and MMP-9 levels in GCF were found in patients with chronic periodontitis and their GCF levels significantly decreased after non-surgical periodontal therapy (Marcaccini et al., 2010). Therefore, MMPs can be considered valuable biomarkers for the detection of periodontitis and its severity, activity, and assessment of therapy outcomes (Checchi et al., 2020).

In periodontal disease, periodontal pathogens induce reactive oxygen species (ROS) production which causes ground substance breakdown. Therefore, antioxidants can be used as an adjunct to periodontal therapy to act as scavengers for ROS reducing the extracellular matrix (ECM) degradation (Hernández-Camacho et al., 2018). Coenzyme Q10 (CoQ10) is currently available as a dietary supplement from 15 to 100 mg (Bhagavan & Chopra, 2006) and is widely recommended as a supplement for cardiovascular diseases (Mortensen et al., 2014 ; Alehagen et al., 2016 ). The use of CoQ10 is having a significant impact in decreasing the production of inflammatory cytokines (Fan et al., 2017). Moreover, CoQ10 may block receptor activators for nuclear factor Kappa (RANKL), thus inhibiting bone resorption, increasing the number of bone trabeculae, and improving bone density (Zheng et al., 2021).

Ubiquinol (the reduced form coenzyme Q10) acts as an endogenous antioxidant which increases the concentration of CoQ10 in the diseased gingiva and decreases the periodontal inflammation (Prakash et al., 2010). The potential value of systemic CoQ10 as an adjunct to non-surgical periodontal therapy has been investigated. This led to a significant decrease in the Gingival and the Sulcus Bleeding index measurements with significant improvement in periodontal pocket depth concluding that coenzyme CoQ10 had an important effect on the periodontal tissue health (Manthena et al., 2015).

However, when CoQ10 was used topically with scaling and root planing, the results were controversial. Sale et al., 2014 confirmed the role of CoQ10 in reducing bleeding on probing in conjunction with non-surgical periodontal therapy, after a follow-up period of 4 weeks. In addition,  Raut et al., 2019 confirmed that subgingival application of CoQ10 has a beneficial role in smokers suffering from periodontitis. On the other side, Sharma et al., 2016 showed that CoQ10 had no added value in decreasing bleeding on probing but enhanced pocket depth reduction.

There are very few studies evaluating the benefit of CoQ10 in periodontal therapy and that it needs more investigations as stated by (Dommisch et al., 2018).  Hence, the present study was performed to assess the effect of Coenzyme Q10 supplementation as an adjunct to mechanical periodontal therapy for the management of periodontitis and its effect on GCF levels of biomarkers such as MMP9.

Subjects and Methods:

I. Patient selection

Thirty two patients had participated in the present randomized controlled clinical trial. They were selected from the clinic of periodontology in the Diagnosis, Oral Medicine and Periodontology Department, Faculty of Dentistry, Cairo University. The study protocol was compliant with the regulatory guidelines of the declaration of Helsinki. All steps of the study were explained in full details to each patient who signed an informed consent. All patients were diagnosed as having Stage II Grade B periodontitis (Caton et al., 2018). Any patient having any systemic conditions that may affect or modify the treatment were excluded from the study. Smokers and pregnant females were also excluded. Any patient who was taking any medication that might affect the outcomes of the study was excluded. All patients were instructed not to take any medications during the 3 months period of the study without informing the operators.

II. Initial therapy

All patients were instructed to maintain strict oral hygiene measures, including brushing with soft tooth brush and use of either interdental brush or dental floss.

Periodontal examination was performed, probing pocket depth (PD) and clinical attachment level (CAL) wererecorded to the nearest millimeter using William’s graduated periodontal probe. Periapical radiographs were also done to confirm the bone boss at the sites of attachment loss.

Initial periodontal therapy for all patients included full mouth ultrasonic and manual supra, subgingival scaling and root planing on two consecutive sessions.

The 32 patients were randomly allocated into 2 groups using a random allocation computer software. Each patient was randomly assigned to either the test or the control group using a computer-generated randomization list with a 1:1 allocation ratio. Allocation concealment was done utilizing numbered opaque sealed envelopes that contained the treatment group to which the participant was assigned (done by A.B.). The test group: patients received coenzyme Q10 30 MG (Arab Company for Pharmaceuticals & Medicinal Plants) (MEPACO-MEDIFOOD) twice daily for 3 months. Nothing was given to the control group.  

Regular visits (each month) were scheduled for each patient to check for the oral hygiene and removal of any supragingival plaque or calculus if present.

Clinical parameters for the selected sites (PD and CAL) were measured preoperatively and 3 months postoperatively.

III. Gingival crevicular fluid (GCF) sampling procedure:

The GCF samples for each patient was collected before scaling and root planing,  1 month and 3 months after the non-surgical periodontal therapy. The selected sites were isolated with cotton rolls, dryness of the area of interest was done and supragingival plaque, if present, was removed using a periodontal probe to prevent saliva and/or plaque contamination.  Pre-cut filter paper strips (3mm x 9 mm) were placed in the crevice until minimal resistance was felt and were left in position for 1 min and then placed in plastic Eppendorf (Fig.1). Any strips with any plaque, saliva or blood contamination were discarded. The samples were frozen at -80° C till use. After collection of the 96 GCF samples, the paper strips were allowed to thaw at room temperature for 30 minutes. Then elution of the GCF samples; by adding 100 μl of phosphate buffered saline (PBS) to each eppendorf and centrifuged at 11,000 r.p.m. for 15 minutes; was done.

Fig. 1:  Filter paper placed inside the periodontal pocket for GCF sample collection 

IV. MMP9 detection and measurement of its GCF level

Assessment of MMP-9 levels in GCF was performed using RayBio^® Human MMP-9 ELISA Kit (RayBiotech Life, Inc. U.S.A.). This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for MMP9 had been  pre-coated onto a microplate. Standards and samples were pipetted into the 96 wells and any MMP9 present was bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for MMP9 was added to the wells to sandwich the MMP9 immobilized during the first incubation. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution was added to the wells and color was developed in proportion to the amount of MMP9 bound in the initial step. The color development was stopped and the color intensity finally was measured in pg/ul using an ELISA reader.

Sample size:

The sample size was estimated based on (Pranam et al, 2020). The sample size for each group was 14 participants. Based on (0.05) α error, (95%) power and effect size of (1.2) with two arms. Sample size was increased by around 15% per each group to compensate for any missing data.

Statistical analysis:

Data were explored for normality using the Shapiro-Wilk test. Continuous variables were described as mean and standard deviation. The within-group comparison was performed using Paired t-test and repeated measure one-way ANOVA test with Geisser-Greenhouse's epsilon correction. The comparison between the two groups was performed using a two-tailed unpaired T-test. All statistical analyses were done using IBM Corp. Released 2020. IBM SPSS Statistics for Windows, Version 27.0. Armonk, NY: IBM Corp. Differences were considered to be significant when the P-value was less than .05.

Results:

The demographic data and baseline values were described in table 1. There was no attrition during the trial. There were no reported side effects.

Table 1: Demographic characteristics and baseline values

Variables are significant when P-values less than .05

Both groups showed a statistically significant decrease in all the measurement parameters compared to the baseline as shown in Tables 2, 3, and 4.

Table 2: Intra-groups change in PD

*Variables are significant when P-values less than .05

Table 3: Intra-groups change in CAL

*Variables are significant when P-values less than .05

Table 4: Intra-groups change in MMP-9 GCF levels

*Variables are significant when P-values less than .05

Means in a column without the common subscript letters significantly differ

A statistically significant difference in PD reduction was detected between the test group and the control group with a p-value of 0.0031. Also a statistically significant difference in CAL gain was detected between the test and the control groups with a p-value of 0.0004.

A statistically significant difference in MMP9 GCF levels decrease was detected between the test and the control groups after 1 month with a p-value of 0.02 but more decrease was detected after 3 months with a p-value < 0.0001 as shown in table 5 and fig.2   

Table 5: Inter-group comparison between different parameters

*Variables are significant when P-values less than .05

Fig. 2: Change in MMP9 CGF levels in both groups throughout the study time intervals

Discussion

The ROS-induced destruction fueled the search for supplementary  anti-oxidants in the treatment of various inflammatory diseases including periodontitis. CoQ10 has been widely used in the medical field, due to its anti-oxidant properties. Various researches showed that  CoQ10may be effective as a topical or systemic adjunctive treatment for periodontitis (Prakash et al., 2010 ; Manthena  et al., 2015 ; Shoukheba & El- Kholy, 2019 ; Pranam et al., 2020 ; Hans et al., 2021 and Sale et al., 2021).

Many studies demonstrated the benefit of using of CoQ10 in the management of periodontal diseases, but very few studies evaluated its effect on the biomarkers besides the clinical parameters and no study evaluated the effect of CoQ10 on MMP-9 GCF levels. Therefore, the rationale behind conducting this study was to assess the benefit of CoQ10 as an adjunct to non-surgical therapy in treating periodontitis and to evaluate its impact on GCF levels of MMP9 which is one of the major matrix metalloproteinases that plays a role in the pathogenesis of periodontal disease. The used CoQ10 dosage, which is recommended by the manufacturing company, was safely applied with same or even longer durations in previous studies related to non surgical periodontal therapy. (Manthena  et al., 2015 ; Shoukheba & El- Kholy, 2019).

The results of the current study showed that there was a statistically significant decrease in the clinical parameters from baseline to 3 months postoperatively in each group separately. This is in accordance with the results of the studies performed by Manthena et al., 2015 ; Sharma et al 2016 and Pranam et al., 2020 who compared the clinical parameters in patients with and without topical use of CoQ10 gel in conjunction with scaling and root planing. They recorded a statistically significant decrease in the probing depth, gingival index with a significant gain in attachment level in each group separately. These results were further confirmed by Raut et al., 2019 who applied CoQ10 topically in smokers with chronic periodontitis and Shoukheba & El- Kholy, 2019 who administered systemic CoQ10 in diabetics with periodontitis. Both studies demonstrated the clinical efficiency of CoQ10 subgingival application in the significant reduction of the gingival inflammation and PD with significant gain in CAL.

In the current study, there was a statistical significant difference in PD reduction, CAL gain between both groups in favor of the group treated with CoQ10 in conjunction with non-surgical periodontal therapy. This is in accordance with the results of the  study conducted by Raut et al., 2019 and Sale et al., 2021  who reported a statistical significant difference in decrease of PD and gain in CAL in the topical CoQ10 with scaling and root planing group compared to scaling and root planing alone after 3 months and 4 weeks in these studies respectively.

Similarly, Shoukheba & El- Kholy, 2019 in a prospective clinical study divided chronic periodontitis patients with Diabetes Mellitus II into two groups, group I was treated with scaling and root planing plus placebo capsules, and group II was treated with scaling and root planing with systemic 30 mg CoQ10. They reported that there was a significant statistical difference in decrease of  PD and gain in CAL  between both groups in favor of the CoQ10 group in all the study time points. The added benefit of CoQ10 to the non-surgical periodontal therapy was recently confirmed by Hans et al 2021.

In contrast to our study, Pranam et al., 2020  compared the clinical parameters between the non-surgical treatment of periodontitis with and without the topical use of CoQ10. They found no statistically significant difference between them in all the clinical parameters concerning the change in PD and CAL. This might be justified by the use of CoQ10 in the form of a gel applied only once, unlike our study which used oral supplementation twice daily for 3 months. They postulated that there was no difference between the 2 groups because of the unconfirmed substantivity of the CoQ10 and the gel retention in the periodontal pocket could be an issue. This preparation was neither sustained nor controlled release which may have not allowed to offer the required effect.

Regarding the GCF MMP9 level in the current study, there was a statistically significant difference decrease in its levels in each group separately whether after 1 or 3 months postoperatively. The decrease in MMP9 levels in GCF in periodontitis patients after scaling and root planing was confirmed by the studies performed by Marancini et al., 2010 and Attia and Alblowi 2020. Moreover, when comparing the change that occurred in MMP9 GCF levels between both groups of the current study, there was a statistical significant difference in favor of the test group after 1 and 3 months.

To the best of our knowledge, there is no study that has investigated the impact of using CoQ10 whether topically or systemically on MMP9 levels in GCF. However, the findings of this study are somehow in agreement with those reported by Shoukheba & El- Kholy, 2019. They reported a statistically significant difference in the MMP8 GCF levels decrease when comparing scaling and root planing with and without the systemic use of CoQ10 in favor of the combination therapy. MMP8 and 9 are two major matrix metalloproteinases that play a role in the destruction of different types of collagen and ECM components (Buzoglu et al., 2009 and de-Paulo Silva et al., 2009). The antioxidant impact of CoQ10 and its inhibitory effect on MMP8/9 GCF levels can be justified by the findings reported by Pranam et al., 2020.  They recorded a significant increase in superoxide dismutase (SOD) enzyme level in GCF after using CoQ10 in addition to the non-surgical treatment when compared to scaling and root planing alone in patients with periodontitis. SOD is considered one of the enzymes that scavenges ROS which are important for the upregulation of MMP8/9 gene expression (Lu and Wahl 2005).

All of these findings confirm the superiority of combined scaling and root planing and CoQ10 over scaling and root planing alone in the management of periodontitis in terms of reduction of PD, gain of CAL, and decrease of MMP-9 GCF levels. However, the statistical significant difference recorded between both groups in the clinical parameters might be of a less clinical significance and can be a bit misleading on the clinical aspect. Hence, further studies are recommended to evaluate the clinical impact of using different doses of CoQ10 in combination with non surgical periodontal therapy. Moreover, more studies are required to evaluate its effect on the non surgical periodontal therapy of more advanced cases such as stage III periodontitis.

Conclusion:

Within the limitations of the present study, as the absence of  participants blinding,  we can conclude that the antioxidant action of CoQ10 offered an added benefit to the non-surgical periodontal therapy in terms of significant gain in CAL, reduction in PD and MMP9 GCF level. 

Conflict of Interest

The authors declare that there is no conflict of interest

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.