Effect of simvastatin on expression of Interleukins 6 & 10 and Matrix Metalloproteinase: 9 when used as an intracanal medicament in teeth with symptomatic apical periodontitis-a triple blind randomized controlled trial

Abstract

Background

The objective of this prospective, triple-blind, randomized clinical controlled trial is to investigate and compare the expressions of interleukin-6 (IL-6), interleukin-10 (IL-10), and matrix metalloproteinase-9 (MMP-9) in the periapical tissues of teeth with symptomatic apical periodontitis following the placement of calcium hydroxide (Ca(OH)₂) and simvastatin as intracanal medicaments (ICM). Additionally, the study aims to evaluate pre-treatment and interappointment pain levels. This research could lead to improved treatment protocols for symptomatic apical periodontitis, enhancing patient comfort and outcomes.

Methods

The study adhered to CONSORT guidelines. A total of 34 patients aged 14 to 60 years with single-rooted teeth diagnosed with pulp necrosis and symptomatic apical periodontitis underwent root canal procedures. They were randomly divided into two groups based on the ICM used: Group I received Ca(OH)₂, while Group II received simvastatin. Tissue fluid samples were collected at three time points (T0, T1, T2) using paper points inserted into the periapical tissues immediately after access opening, after cleaning and shaping, and 7 days after ICM placement. The samples were stored at – 20 °C for analysis. Pain levels were recorded using the Heft Parker Visual Analogue Scale (HP-VAS) at various intervals. All samples underwent enzyme-linked immunosorbent assay (ELISA) to estimate IL-6, IL-10, and MMP-9 levels in pg/ml. Statistical analysis included the Mann–Whitney U test and Quade nonparametric ANCOVA for inter-group comparisons, while intra-group comparisons were performed using the Friedman test.

Results

Statistically significant differences were observed in IL-6 and MMP-9 levels within groups but not in IL-10 across time points (p < 0.05). In intergroup comparisons at T2, simvastatin showed significantly lower expressions of IL-6 and MMP-9 compared to Ca(OH)₂ (p < 0.05). IL-10 levels increased in both groups without significant differences. Pain scores were significantly lower following simvastatin treatment compared to Ca(OH)₂ (p < 0.05).

Conclusions

The expression of these biomarkers indicates that simvastatin is effective in reducing inflammation and pain in teeth with pulpal necrosis and symptomatic apical periodontitis when used as an intracanal medicament compared to Ca(OH)₂.

Trail Registration: Clinical Trial Registry of India CTRI/2022/08/044749; Registered 18 August 2022 https://drive.google.com/file/d/17JXArM3qoqvTMiUa9ITMTQTGLEvsxpW_/view?usp=sharing

Background

Apical periodontitis (AP) is an inflammatory condition that arises from bacterial infection in the necrotic root canal system. This infection triggers a host inflammatory response, characterized by the release of virulence factors, toxins, and inflammatory mediators from pathogenic species within the infected root canals. These substances infiltrate the periapical tissues, initiating an inflammatory process that can lead to bone resorption around the tooth apex [1, 2]. In response to this infection, immunocompetent cells migrate to the periapical area, where they release pro-inflammatory cytokines such as interleukin-6 (IL-6), interleukin-1 beta (IL-1ß), and tumor necrosis factor-alpha (TNF-α), along with anti-inflammatory cytokines like interleukin-10 (IL-10) and interleukin-4 (IL-4). These cytokines play crucial roles in modulating inflammation and tissue repair [2, 3]. Cytokines are small signaling proteins that serve as important markers of inflammation. For instance, IL-6 is rapidly released at the infection site to combat pathogens, while IL-10 functions as a potent anti-inflammatory agent that inhibits pro-inflammatory cytokines, thereby promoting healing. Moreover, matrix metalloproteinases (MMPs) are involved in the degradation of the extracellular matrix (ECM) and bony matrix in the periapical region. Research has shown elevated expression of MMP-9 in periapical lesions, indicating its significant role in the progression of these lesions [3]. Although chemo-mechanical debridement effectively reduces bacterial load and inflammatory markers, it does not eliminate them. Consequently, the placement of intracanal medicaments (ICMs) may become essential to manage persistent infections effectively [4]. This underscores the need for comprehensive treatment strategies in managing apical periodontitis to ensure successful outcomes.

Statins, specifically 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG CoA) reductase inhibitors, are primarily known for their cholesterol-lowering capabilities. However, they also exhibit significant immunomodulatory, antioxidative, and anti-inflammatory properties. Research by Sakoda et al. demonstrated that simvastatin has an anti-inflammatory effect on human oral epithelial cells, leading to a reduction in the production of interleukin-6 (IL-6) and interleukin-8 (IL-8).Various methodological approaches—including in vitro studies, animal models, observational studies, randomized clinical trials, and meta-analyses—have consistently affirmed the effectiveness of simvastatin in managing chronic periodontitis. This effectiveness is noted for both local and systemic applications of the drug. These findings highlight the potential of simvastatin as a therapeutic agent not only for hyperlipidemia but also for inflammatory conditions such as chronic periodontitis [5].

Numerous animal model studies and clinical trials have investigated the effects of statins on bone formation, particularly in the context of osseointegration of implants and local applications in extraction sites to prevent alveolar bone resorption [6]. Research indicates that local application of statins yields significantly more beneficial effects on dental and oral health compared to systemic administration. In addition to their anti-inflammatory properties, recent evidence suggests that statins enhance gene expression of bone morphogenic protein-2 (BMP-2) and inhibit matrix metalloproteinases (MMPs), which stimulates the differentiation of osteoblastic bone marrow stem cells and promotes wound healing [7]. This rationale supports the use of statins via local administration due to their improved bioavailability in targeted areas. However, there is currently a lack of clinical studies evaluating the efficacy of simvastatin as an intracanal medicament. The primary objective remains to create an environment conducive to the healing of periapical tissues. Therefore, this triple-blind, randomized clinical study aims to assess the immunological status of periapical infections by evaluating the ratios of IL-6 and IL-10 cytokines, as well as MMP-9 levels, following the placement of simvastatin as an intracanal medicament in infected root canals of teeth with symptomatic apical periodontitis. Additionally, the study seeks to investigate potential correlations between biomarker levels, pain, and inflammation in the periapex. The null hypothesis posits that there will be no significant differences in biomarker expression or pain levels between the simvastatin group and the control group.

Methods

Source of data, selection criteria, sample size calculation

This randomized clinical trial has been written according to Preferred Reporting Items for Randomized Trial in Endodontics (PRIRATE) 2020 guidelines (Fig. 1) [8] and CONSORT Guidelines 2010. The protocol of this prospective, triple-blind, single-center, randomized, clinical trial was designed in accordance with the World Medical Association Declaration of Helsinki. The study was approved by Institutional Review Board (SRMDC/IRB/2020/MDS/No.307) and registered with Clinical Trial Registry of India CTRI/2022/08/044749; Registered 18 August 2022 https://drive.google.com/file/d/17JXArM3qoqvTMiUa9ITMTQTGLEvsxpW_/view?usp=sharing Based on effect size of the previous study [9] comparing the MMP levels following Ca(OH)₂ placement, the sample size calculation was done with alpha err prob 0.05 and power (1-ß err prob) 0.80. The sample size calculations (n = 34) were done using software programme (SPSS software version 23.0).

Fig. 1.

PRIRATE 2020 Flow Chart

Systemically healthy forty patients who reported to the outpatient section to the Department of Conservative Dentistry for root canal treatment were screened and assessed for eligibility based on the inclusion and exclusion criteria, of which 6 were excluded,4 were not meeting the inclusion criteria and 2 declined to participate. Remaining 34 were recruited and explained about the procedure after taking the patient consent form (2020–2023).

The following inclusion criteria were adopted to standardize the study protocol; systemically healthy patients aged 18–60, patients who required root canal treatment of a single rooted, single canaled, mature teeth diagnosed with pulp necrosis and symptomatic apical periodontitis. Clinical examination of patients included medical and dental history followed by the elaborate history of the chief complaint. Intraoral and extraoral clinical examination of the involved tooth were achieved accurately using a diagnostic mirror and probe. To assess the pulpal status of the involved teeth, pulp testing with cold testing (ethyl chloride spray) and electric pulp tester was used. The contralateral and the tooth of interest was examined. Prolonged sharp intense painful response to cold and electric pulp testing was considered a criterion for vital pulp, while no response was confirmed as the presence of a nonvital pulp or pulpal necrosis. Periapical status was evaluated with clinical examination and periapical radiographs with different angulations. The diagnosis of symptomatic apical periodontitis was confirmed with the chief complaint of pain on biting, responding positively to the percussion test and with a periapical index (PAI) score of 3 (changes in bone structure with some mineral loss, characteristic of apical periodontitis) [10]. Exclusion criteria included patients under any medication for a week or under ASA 2–5 category. Teeth with cracks, vertical root fracture, internal & external resorption, or single rooted teeth with multiple canals or immature apex, dental anomaly, sinus tract were also excluded from the study. Enrolled patients were explained about the study protocol in the vernacular language and informed consent was obtained from the patients willing to participate in the clinical trial. In order to record the pain, patients were explained about how to interpret Heft Parker visual analogue scale (HP-VAS) [which has a millimeter (mm) demarcation based on the intensity of pain] by one of the operator before randomization and preoperative pain score was noted using HP-VAS.

Randomization, clinical and sampling procedures

The participants were then randomly assigned to two groups (I & II) (n = 17) using simple randomization. Participants are allocated to groups by using random number generator software (Randomizer.at). Patients were blinded to their assigned group throughout the study.

Operator 1, who was blinded to both the groups, performed the endodontic procedure. Firstly, antisepsis of the oral cavity was done by using 1% povidone iodine, followed by supra-gingival scaling and cleansing with pumice. Local anesthesia was administered using 2% lignocaine hydrochloride with epinephrine 1:80,000. Following rubber dam isolation, caries was removed using round bur. The crown and surrounding structures were then disinfected with 30% H₂O₂ for 30 s, followed by 3% NaOCl for 30 s, after which it was inactivated with 5% sodium thiosulfate and flushed with sterile saline. Access cavity was prepared using a sterile size 2 Endo access, (Endo Z bur, Dentsply Maileffer, Switzerland) to create a straight line access. Glide path was established with #10 k-file and then with #15 k-file and working length was determined using an electronic apex locator (Root ZX Morita Corporation, Kyoto, Japan). After the canal was completely dried, operator 2, who was unaware of the treatment protocol collected the samples (T₀), by introducing the paper point tip which was standardised as 15/0.02. (Dai Pro T paper points, Diadent group international, Korea, Lot no:010522) into the canal extending 2 mm beyond the apex (based on the predetermined working length) and retained in place for 60 s to obtain periapical tissue fluid samples. A wet paper point indicates that it has been introduced into the periapical tissue, confirming the extent of insertion. Paper points were immediately transferred to cryo-vial tubes containing phosphate buffered saline to be stored in a freezer at – 20 °C.

Following cleaning and shaping using ProTaper Gold files (Dentsply Sirona, USA) up to F3 with crown down technique and intermittent irrigation with continuous chelation [3% NaOCl + 9% HEBP] using 29-gauge side vented needle 1 mm short of working length, it was neutralized by 5% sodium thiosulfate, followed by a final flush with 2 ml of saline. Recapitulation was performed by # 10 K-file to maintain the patency. Root canals was dried using paper points. After the canals was completely dried, post preparation sample (T₁) was collected by introducing the paper point tip which was standardised as 15/0.02. (Dai Pro T paper points, Diadent group international, Korea, Lot no:010522) into the canal extending 2 mm beyond the apex (based on the predetermined working length) and retained in place for 60 s to obtain periapical tissue fluid samples and intracanal medicaments were placed based on the randomization. All the procedure was done under dental operating microscope. (LABO AMERICA INC, USA; PRIMA DNT Surgical Microscope)

Group I(CH): Ca(OH)₂ powder mixed with methylcellulose in a ratio of 1 g/0.8 ml.

Group II(SIM): Simvastatin powder mixed with methylcellulose in a ratio of 1.2 g/100 ml.

Formulation of 1.2% simvastatin paste

Despite the documentation of statins anti-inflammatory properties, use of high doses have been reported to cause soft tissue inflammation. Hence accordingly, the dosage of simvastatin was kept to 1.2 mg [11–13]. Literature evidences that 1.2 mg reduces inflammation without compromising the bone growth potential compared to 2 mg.

In situ methylcellulose gel was prepared similar to Thylin et al. [14]. Shortly, methylcellulose was prepared by adding the required quantity of biocompatible solvent to the vial containing precisely weighed amount of methylcellulose. The vial was heated to about 50 °C to 60 °C and with the use of mechanical shaker it was agitated to obtain a clear solution. The required quantity of simvastatin was added to the vial containing the solution and was completely dissolved to obtain a homogeneous phase gel of 1.2% simvastatin.

Simvastatin as ICM paste

For the purpose of standardization, 4–5 ml of the prepared simvastatin gel was delivered into the canal based on the size using a lentulo spiral in a slow-speed handpiece and tamped down in the canal space using the blunt ends of sterile paper points. Due to the limitation of simvastatin being not opaque, radiographs were not considered to assess its extent of placement. The entire procedure was done under dental operating microscope and the extent of its placement was assessed through microscope.

Similarly Ca(OH)₂ was mixed to obtain a desired consistency and was placed in the canal as same as the placement of simvastatin. Coronal access was restored with temporary material Cavit-G (3 M ESPE,USA). Patients were monitored for pain at various intervals following placement of intracanal medicaments (6-,12-,18-h and 1st day, 2nd day,3rd day, 4th day, 5th day 6th day and on 7th day) using Heft Parker visual analogue scale (HP-VAS). Patient were recalled after 7 days, at which time, under rubber dam isolation and similar aseptic conditions as mentioned above, the coronal seal was opened, the ICM was removed using sterile saline irrigation under/with ultrasonic agitation. The canal was dried using paper points and sample collection (T₂) was done with the standardized paper point tip 15/0.02. (Dai Pro T paper points, Diadent group international, Korea, Lot no:010522). It was inserted similarly 2 mm beyond the working length and was retained in place for 60 s to obtain periapical tissue fluid samples. Paper points with spot bleeding were discarded and not used for the study. The obturation was then completed using lateral condensation and post endodontic restoration done with composite resin. Following the completion of endodontic procedure, patients were recalled at 1,3 and 6 months and the pain scores were again evaluated by the operator who was blinded, using HP VAS. If pain persisted, thorough reexamination was planned to analyze for flare up, missed canals, fracture or neuropathic pain. But none reported with prolonged pain.

Estimation of cytokine level

Levels of IL-6, IL-10, and MMP-9 were determined using commercially available ELISA kits (Abbkine Scientific Co, USA). Briefly, 50µL of the cell culture supernatant was added to the appropriate wells of the ELISArray plate, which included all specific cytokines/chemokine capture antibodies. After 2 h of incubation at room temperature, the plate was washed, and 100 µL of detection antibody was added to the appropriate wells and incubated at room temperature. Subsequently, 100 µl of diluted avidin-horseradish peroxidase (HRP) was added to the appropriate wells and incubated for 1 h at RT for 30 h. After washing, 100 µl of the development solution was added to each well and incubated for 15 min at RT in the dark. The samples were rinsed once more with distilled water after an hour at room temperature, and were then examined under a micro-ELISA reader at 450 nm to determine the amount of IL-6, IL-10 and MMP-9 expression in pg/ml. The samples were analyzed in triplicates (number of patients-34;sample per patient-3;total sample-102; triplicates-306).The values obtained were subjected to statistics, which was blinded throughout the analysis.

Statistical analysis

SPSS software version 23.0 was used for statistical analysis. Shapiro–Wilk test was used to assess whether the data obtained for was normally distributed. Data was not found to be normally distributed. Hence, non-parametric tests were conducted using Mann Whitney U test and Quade nonparametric ANCOVA for inter-group comparisons for the expression of biomarkers. Intra-group comparison for expression of biomarkers were done using Friedman test. The level of significance was set at p < 0.05. Intergroup comparison for HP-VAS scores (pre-operative and post-operative recorded at 6-,12-,18-h.and 1st day, 2nd day,3rd day, 4th day, 5th day 6th day, 7th day,1,3 and 6 months were performed using Mann–Whitney U test. p < 0.05 was considered to be statistically significant.

Results

Thirty four patients who reported with pulpal necrosis and symptomatic apical periodontitis were monitored at different time periods (T₀,T₁,T₂) using CH and SIM as ICM to analyze the IL-6, IL-10 and MMP-9 levels. There was no loss of follow-up reported. Table 1 represents baseline data with mean distribution of gender, age and preoperative pain scores in CH and SIM group. Table 2 presents the descriptive data of IL-6,IL-10,MMP-9 (pg/ml) for CH and SIM at different time periods. Table 3 represents the mean HP-VAS scores for the assessment of post-operative pain in CH and SIM group at 6-,12-,18-h post operatively and 1st,2nd,3nd,4th,5th,6th and 7th day, 1,3 and 6 months. Figures 2, 3 and 4 depicts the IL-6,IL-10 and MMP-9 values respectively at various time period for CH and SIM.

Table 1.

Baseline data with mean distribution of gender, age and preoperative pain scores in CH and SIM group

Baseline Parameters	Group I CH	Group II SIM	P value
Gender M/F(n= 34)	10/7	9/8
Age (years)	45.16± 9.96	38.7± 11.14	0.11
Preoperative pain score (mm)	127± 20	136± 17	0.16

Table 2.

Descriptive statistics of IL-6, IL-10, MMP-9 values (pg/ml) for CH and SIM at various time intervals

		IL-6 CH SIM		IL-10 CH SIM		MMP-9 CH SIM	P value
T0	Mean	40.61	50.91a	2.61	3.56	269.11a 263.54
	Std. dev	9.46	14.23	0.39	0.37	21.42 4.05
	Std. error	2.29	3.46	0.24	0.24	5.20 0.98	0.05
T1	Mean	25.81a	29.22a	3.17	3.91	229.21a 169.26a
	Std. dev	3.83	7.01	0.53	0.53	25.58 2.90
	Std. error	0.93	1.70	0.33	0.33	6.22 0.71	0.05
T2	Mean	20.70a	16.76a	4.01	5.61	157.74a 132.23a
	Std. dev	2.93	2.95	0.89	1.88	15.71 2.99
	Std. error	0.72	0.72	0.93	0.93	3.82 0.73	0.05

^{a=Denotes statistically insignificant}

Within the same row, values with the same letters were not significantly different at p = 0.05

T₀—Samples following access opening

T₁—Samples following cleaning and shaping

T₂—Samples following placement of ICM

Table 3.

Interappointment and post-operative mean HP-VAS scores experienced in patients with CH and SIM as intracanal medicament at various time intervals

Time interval	Group	N	Mean	Std. deviation	Std. error mean		Median	Inter-quartile range	p value
6 h	CH	17	107.94	11.638	2.823		106	19	<0.001**
	SIM	17	91.59	7.500	1.819		91	12
12 h	CH	17	89.71	9.252	2.244	90		15	<0.001**
	SIM	17	68.88	10.093	2.448	68		13
18 h	CH	17	75.24	11.361	2.755	74		17	<0.001**
	SIM	17	42.12	10.234	2.482	40		10
1st day	CH	17	59.29	9.413	2.283	60		14	<0.001**
	SIM	17	23.24	8.408	2.039	21		11
2nd day	CH	17	35.35	7.254	1.759	35		14	<0.001**
	SIM	17	13.53	9.735	2.361	16		21
3rd day	CH	17	14.41	7.722	1.873	15		8	<0.001**
	SIM	17	2.53	3.145	0.763	0		5
4th day	CH	17	4.00	3.606	0.874	4		7	<0.001**
	SIM	17	0.00	0.000	0.000	0		0
5th day	CH	17	0.00	0.000a	0.000	0		0	1.000
	SIM	17	0.00	0.000a	0.000	0		0
6th day	CH	17	0.00	0.000a	0.000	0		0	1.000
	SIM	17	0.00	0.000a	0.000	0		0
7th day	CH	17	0.00	0.000a	0.000	0		0	1.000
	SIM	17	0.00	0.000a	0.000	0		0
1 month	CH	17	0.00	0.000a	0.000	0		0	1.000
	SIM	17	0.00	0.000a	0.000	0		0
3rd month	CH	17	0.00	0.000a	0.000	0		0	1.000
	SIM	17	0.00	0.000a	0.000	0		0
6th month	CH	17	0.00	0.000a	0.000	0		0	1.000
	SIM	17	0.00	0.000a	0.000	0		0

^** denotes statistically highly significant (statistically analyzed using Mann–Whitney U Test)

Heft Parker visual analog scale = 0–170 mm (scoring criteria: < 0 = no pain, > 0 ≤ 23 = faint pain, > 23 ≤ 36 = weak, > 36 ≤ 54 = mild pain, > 54 ≤ 85 = moderate, > 85 ≤ 114 = strong, > 114 ≤ 170 = intense pain, > 170 = maximum possible

Fig. 2.

The mean IL -6 values (pg/ml) for CH and SIM at various time intervals

Fig. 3.

The mean IL-10 values (pg/ml) for CH and SIM at various time intervals

Fig. 4.

The mean MMP-9 values (pg/ml) for CH and SIM at various time intervals

IL-6 observation

According to the results of our study, in CH group, the mean IL-6 levels was 40.61 ± 9.46 pg/ml at the time period T₀. Following biomechanical preparation (T₁), the IL-6 levels reduced to 25.81 ± 3.83 pg/ml and further lowered to 20.70 ± 2.93 pg/ml after 7 days of CH placement (T₂). Similarly, in SIM, mean IL-6 levels at T₀ was found to be 50.91 ± 14.23 pg/ml that decreased to 29.22 ± 7.01 pg/ml and 16.76 ± 2.95 pg/ml at T₁ and T₂ respectively.

Intragroup analysis of interleukin-6 (IL-6) levels at time points T₀, T₁, and T₂ showed statistically significant differences between each time period. This holds good for both the CH and SIM groups, indicating that IL-6 levels changed significantly after the placement of ICMs.

At T₀, there was a significant difference in IL-6 levels between the two groups. This difference could have influenced the results, so it was taken into account as a covariate in further analysis.

Intergroup comparisons at T₁ revealed no statistically significant differences, indicating similar reductions in both the groups following biomechanical preparation. At T₂, IL-6 levels were significantly lower in the SIM group than in the CH group (p < 0.05), demonstrating the superior efficacy of simvastatin over the same time period.

IL-10 observation

In this study the mean IL-10 levels in the CH group were 2.61 ± 0.39 pg/ml at time point T₀. After biomechanical preparation (T₁), IL-10 levels elevated to 3.17 ± 0.53 pg/ml and further to 4.01 ± 0.89 pg/ml after 7 days of CH placement (T₂). Similarly, mean IL-10 levels in the SIM group were 3.56 ± 0.37 pg/ml at T₀ and went up to 3.91 ± 0.53 pg/ml and 5.61 ± 1.88 pg/ml at T₁ and T₂, respectively.

Intragroup analysis of IL-10 levels at time points T₀, T₁, and T₂ showed no statistically significant differences between time periods in either the CH or SIM group. This suggests that IL-10 levels were elevated after ICM placement.

Baseline IL-10 levels differed significantly between the groups, so this was controlled for in subsequent analyses. Biomechanical preparation and ICM placement resulted in similar reductions in IL-10 levels in both the groups at T₁ and T₂, with no significant difference between the two groups.

MMP-9 analysis

Mean MMP-9 levels in the CH group were 269.11 ± 21.42 pg/ml at baseline (T₀), decreasing to 229.21 ± 25.58 pg/ml after biomechanical preparation (T₁) and 157.54 ± 15.71 pg/ml after 7 days of CH placement (T₂). Similarly, in the SIM group, mean MMP-9 levels were 263.54 ± 4.05 pg/ml at baseline (T₀), decreasing to 169.26 ± 2.90 pg/ml at T₁ and 132.23 ± 2.99 pg/ml at T₂.

Intragroup analysis showed statistically significant difference in MMP-9 levels between each time point (p < 0.05).

Intergroup comparisons at T₂ revealed statistically significant differences in MMP-9 levels, among the groups at T₂, and MMP-9 levels were significantly lower in the SIM group than in the CH group (p < 0.05).

Intergroup comparison for HP VAS score (pre-operative and post-operative) revealed that there was statistically highly significant difference between post-operative pain recorded at 6-, 12-, 18 h, 1st day, 2nd day, 3rd day, 4th day(p < 0.001) (Table 3). Lower pain scores were observed for SIM recorded at 6 h (91.59 ± 7.5), 12 h (68.88 ± 10.09), 18 h (42.12 ± 10.23), 1st day (23.24 ± 8.41), 2nd day (13.53 ± 9.73), 3rd day (2.53 ± 3.14) (Table 3). There was an absence of pain for SIM, recorded at 4th day. There was an absence of pain for both the groups recorded at 5th day,6th day and 7th day. There was absence of pain at 1,3 and 6 month follow up (Fig. 5).

Fig. 5.

Bar graph for HP-VAS score (6 h, 12 h, 18 h, 1st day, 2nd day, 3rd day, 4th day, 5th day,6th 7th day,1, 3 and 6 months) for CH and SIM

Discussion

Recognizing a novel material that prevents the progression of periapical lesions might bring about a conducive environment for healing. Intracanal medicaments diminish the root canal bacterial load thereby invoking the healing process. A cascade of several events takes place during this process, namely inflammation, proliferation and remodeling, each distinguished by the interplay of interleukins, cells, growth factors and extracellular matrix [15]. Due to the complex root morphology that include accessory and lateral canals, chemo mechanical preparation with irrigants alone may not be sufficient to completely remove the irritants especially in cases of pulp necrosis with periapical lesions. In these situations, ICM is warranted, particularly for resistant infections [16]. In this regard, the present study quantified and compared the expression levels of IL-6, IL-10 and MMP 9 before and after cleaning and shaping and 7 days following the placement of simvastatin and Ca(OH)₂ as ICM in teeth with pulpal necrosis and symptomatic apical periodontitis with PAI score 3 using ELISA.

IL-6 is a pleiotropic cytokine, and also a critical mediator in inflammatory diseases. IL-6 expression is typically detected only during the acute phase of inflammation, consequently changing the leukocyte infiltration from neutrophils to monocytes and macrophages [17]. IL-10 and MMP-9 have also been recognized for their potential as targets in controlling inflammation. IL-10 is an anti-inflammatory cytokine that play a crucial role in regulating the immune response and suppressing inflammation [18]. On the other hand MMP-9 is an enzyme involved in tissue remodeling and inflammation [19]. Targeting these molecules could offer promising strategies for modulating inflammatory processes and managing related conditions.

Results of intragroup analysis of the IL-6 values showed that chemo mechanical procedures significantly reduced IL-6 levels and were comparable among the groups. It authenticates that the chemo-mechanical preparation of the root canals is capable of reducing the bacterial load and inflammation to a significant level as proven by earlier studies. This finding is in agreement with the previous study by Emad et al. [20]. The IL-6 levels further reduced significantly post ICM placement in both the groups, with significant difference among T₂ levels on intergroup comparison (p < 0.05).

Simvastatin when placed as an ICM was able to reduce the IL-6 levels significantly greater than Ca(OH)₂ during the same time period. This could be ascribed to the anti -inflammatory property i.e. direct effect of simvastatin on IL-6 and C reactive proteins [5]. Simvastatin blocks the intracellular Raf -MEK-ERK pathway, which is one of the well-known intracellular routes that activates the transcript ion factor NF-ĸB, which in turn activates monocytes and macrophages [21]. The antioxidant properties of simvastatin may also have played a role in the decreased expression of IL-6. It reduces oxidative stress by promoting autophagy and repressing osteoblastic apoptosis [22].

Increase in IL-10 levels were observed following chemo mechanical preparation and following ICM placement in both the groups. Several studies have also corroborated the downregulation of inflammation by the use of statins, as demonstrated by increased IL-10 level in gingival crevicular fluid (GCF) from hyperlipidemic patients and in periodontal wound healing [23]. It is been claimed that simvastatin favors IL-10 secretion thereby mitigating the damages caused due to excessive inflammation. Simvastatin reduces the production of pro-inflammatory cytokines like IL-6 and IL-8 in various cell types, including monocytes and endothelial cells. This reduction could create an environment where anti-inflammatory cytokines like IL-10 are more prominent or favored, indirectly affecting IL-10 levels in T cells [24]. IL-10 inhibits bone resorption by arresting the RANK/RANKL pathway [25]. Additionally, it suppresses mRNA expression of cathepsin K a key maker of osteoclast differentiation by inhibiting Src signaling and modulation of MAPK including ERK1/ERK2 [26]. Hence simvastatin can be a potential therapeutic agent to reduce the inflammation-mediated bone resorption.

Expression of MMP-9, also known as gelatinase B is one of the potential biomarkers of inflammation. Once released, it activates cytokines and enhances inflammation and it cleaves and degrades several extracellular matrix (EMC) proteins mainly collagen, causing cell death and tissue loss [27]. It also has several inflammatory response elements, including specificity protein-1, activator protein-1 and NB-_K B sites, which makes it highly responsive to inflammatory stimuli [28].

Statins have been reported to potentially inhibit the expression of MMP-9 which is upregulated by LPS. It has been reported that some protective mechanisms of simvastatin is mediated by impeding the production of MMP-9 [24]. Hence it modulates MMP-9 and prevents its up-regulation, concurrent with the observations of the present study.

While both treatments provided equivalent pain relief after 4 days, simvastatin demonstrates additional therapeutic advantages over calcium hydroxide. Simvastatin accelerates wound healing by inhibiting leukocyte adhesion and migration to inflamed sites, reducing inflammatory cytokine production and T-cell activation. It also enhances macrophage recruitment, which stimulates the proliferation of fibroblasts, keratinocytes, and endothelial cells, and promotes angiogenesis through increased vascular endothelial growth factor (VEGF) production [29]. Furthermore, simvastatin exerts anabolic effects on bone metabolism by upregulating bone morphogenic protein-2 (BMP-2) to enhance osteoblast differentiation, inhibiting osteoblast apoptosis, and suppressing osteoclast function during high bone turnover states. Additionally, it preserves tissue integrity by inhibiting matrix metalloproteinases (MMPs), which prevents extracellular matrix degradation, and improves epithelization processes. These multifaceted benefits make simvastatin a potentially superior choice for conditions requiring both inflammation control and tissue regeneration [30].

On the other hand, calcium hydroxide (CH) lacks angiogenic properties critical for tissue regeneration, its alkaline nature can degrade radicular dentin’s collagen structure through pH-induced mechanical changes, as first reported by Andreasen et al. [31]. In contrast, studies demonstrate that simvastatin application either alone or combined with other materials preserves dentin's fracture resistance, avoiding the structural compromises associated with CH. This distinction positions simvastatin as a mechanically stable alternative that bypasses CH's collagen degradation risks while supporting tissue repair through angiogenic pathways absent in CH-based treatments [5].

To mitigate the influence of the Hawthorne effect on pain assessments i.e. the mere awareness of participants in an investigation can alter the way in which a person behaves, the participants were informed of the study’s objective after they self-recorded their pain levels. By addressing these factors, the integrity of pain assessments was preserved, leading to more reliable outcomes [32–34].

In the current randomized controlled trial, both groups exhibited a gradual and significant decrease in interappointment pain scores over varying time intervals. Concurrently, levels of IL-6 and MMP-9 showed a decline, while IL-10 concentrations increased in both groups throughout the testing period. Notably, a significant difference in cytokine expression and pain levels was observed when simvastatin was used as an intracanal medicament (ICM) compared to calcium hydroxide [Ca(OH)₂]. These results validate the relationship between cytokine expression and pain levels.

Conclusions

The impact of simvastatin in the context of inflammation is favorable, unveiling its potential role as a therapeutic when used as an ICM. Within the limitations of this randomized clinical trial, the results demonstrates that the local release appears to be an agreeably fair solution to the problem with minimalistic side effects. Simvastatin decreased IL-6 and MMP-9 and increased IL-10 levels with significant difference with baseline values and the control group. Significantly less pain was observed in patients with simvastatin compared to Ca(OH)_2.

Abbreviations

Ca(OH)₂

Calcium hydroxide

ICM

Intra canal medicament

ECM

Extra cellular matrix

GCF

Gingival crevicular fluid

LPS

Lipopolysaccharide

RANK/RANKL

Receptor Activator of Nuclear Factor-ĸ beta and Receptor Activator of Nuclear Factor-ĸ Beta Ligand

IL-6

Interleukin 6

IL-10

Interleukin 10

MMP-9

Matrix metalloproteinases

ERK1/ERK2

Extracellular signal-Regulated Kinases 1 and 2

MAPK

Mitogen-Activated Protein Kinase

NF-ĸB

Nuclear Factor- Kappa B

HRP

Horseradish peroxidase

HP-VAS

Heft-Parker visual analogue scale

Author contributions

NK.RK.KS.AS and MS. wrote the main manuscript text and NK.KS.AS. prepared and interpreted all the figures. All the authors reviewed the manuscript and to have agreed to be personally accountable for the authors own contributions. All authors read and approved the final manuscript.

Funding

The research was not funded by any organization. It was self-financed.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author.

Declarations

Ethics approval and consent to participate

The research protocol was presented to Institutional Review Board (IRB) and ethical approval was obtained SRMDC/IRB/2020/MDS/No.307.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing.