The role of systemic statins in the inception and healing of apical periodontitis: a systematic review

The search was conducted independently by two reviewers (FI and MFM). The following electronic databases were searched: PubMed (https://​www.​ncbi.​nlm.​nih.​gov/​pubmed), Scopus (https://​www.​scopus.​com), and Web of Science (https://​www.​webofscience.​com/​wos/​woscc/​basic-search) from the date of foundation of the databases to February, 20 2023. Supplemental research was performed by screening the reference sections of the relevant studies eligible for inclusion in the present systematic review. Furthermore, some digital repositories available such as Google Scholar™ (first one hundred results were considered) and OpenGrey (http://​www.​opengrey.​eu) were investigated in order to explore any conference papers, unpublished data and grey publications. In addition, other resources, including the citations and reference lists of relevant articles and textbooks, were searched manually.

The following keywords were used for research:

Articles found from the above search strategy, after removing duplicates, were first screened by two calibrated reviewers (FI and MFM) based on the relevance of the title and abstract, and sequentially excluded according to the eligibility criteria. Prior to the formal screening process, a calibration exercise was undertaken to pilot and refine the screening questions. To obtain an agreement between the authors, 10% of the publications were randomly selected and their classification was compared, and then a Kappa statistic was determined (Kappa = 0.80). During the second screening, full-text articles were reviewed to make the final inclusion/exclusion decision. In cases of disagreement, a consensus was obtained through discussion or by involving a third reviewer (EC). Articles that fulfilled all the criteria after reading the full text were selected for detailed data processing.

Relevant appraisal tools mentioned in the literature were used to classify the level of information in the articles and assess the risk of bias. The quality appraisal checklists were elaborated on a system where each item was assigned 0 if the item was not fulfilled, 0.5 if partially fulfilled, or 1 if completely fulfilled, and scored independently by two reviewers (FI and MFM). The differences were resolved through discussion. In cases of disagreement, a third reviewer (EC) was involved for reaching a consensus.

Animal studies were evaluated according to the Animal Research Reporting of In Vivo Experiments (ARRIVE) guidelines [42]. Individual full texts were assessed; articles scoring ≤ 5 were classified as low-level, articles with a score between 5 and 15 as moderate, and articles with a score ≥ 15 as high-level information.

The only cohort study included was evaluated using the “Newcastle–Ottawa Quality Assessment Form for Cohort Studies” guidelines [43]. In the evaluation of the full texts, articles with scores less than or equal to 2 were classified as low-level information, scores between 2 and 7 as moderate-level, scores greater than or equal to 7 as high-level information.

The Systematic Review Center for Laboratory Animal Experimentation (SYRCLE) RoB tool was used to assess the risk of bias in animal intervention studies [44]. Eligible articles were evaluated and studies scoring ≤ 3 were classified as having a high risk of bias, those scoring between 3 and 7 as having a moderate risk of bias, and those scoring ≥ 7 as having a low risk of bias.

The “Tool to assess risk of bias in cohort studies of CLARITY Group” was used to grade the risk of bias for the only cohort study included (https://​www.​evidencepartners​.​com/​wp-content/​uploads/​2017/​09/​Tool-to-Assess-Risk-of-Bias-in-Cohort-Studies.​pdf). Among the eligible articles, those scoring ≤ 2 were classified as high risk, those scoring between 2.5 and 6.5 as moderate risk, and those scoring ≥ 7 as low risk of bias.

Data extraction was carried out independently by two reviewers (MFM and FI), using a specially designed form, and the accuracy of the data collected was confirmed by a third reviewer (EC). The following information was extracted: title of the article, authors and year of publication, animal species, samples, disease induction, statins considered, statin mode of administration, treatment period, follow-up, outcome measurements and methods, results and source of funding.

Substantial heterogeneity of methods emerged from the selection of the studies, which precludes the conduct of quantitative data synthesis necessary for a meta-analysis. The clinical results were extracted and qualitatively summarized and a descriptive systematic review was performed.

The searches of PubMed, Scopus, and Web of Science yielded 429, 126, and 156 articles, respectively, for a total of 711 records. Eighty-three articles were left after removal of 628 duplicates. After the initial screening (title and abstract), 76 articles were excluded. The seven articles left were subjected to a full-text review for eligibility assessment [18, 22‐26, 45]. One article was excluded because it did not satisfy the inclusion criteria [40]. Finally, six articles were used for the systematic review [18, 22‐26] (Fig. 1).

The following exclusion criteria were: [1] Studies where participants were not affected by AP, [2] ex vivo, in vitro, and in silico models only studies, [3] studies that included experimental models with comorbidities or with administration of other medications, [4] studies that did not provide a detailed description of the methodology used, [5] review papers, [6] opinion articles and [7] conference abstracts were excluded.

The six in vivo studies selected were published between 2009 and 2018 [18, 22‐26]. Among the included articles, only one was a retrospective human cohort study [18]. The present cohort study analyzed whether there was an association between statin intake and AP healing following NsRCT. All patients included in this investigation (30 cases and 30 controls) received a well-performed initial NsRCT or re-treatment in teeth presenting with AP lesions with a minimum diameter of 3 mm. The treatment was performed by the endodontic residences in a university setting from 2011 to 2014. Patients who took therapies that can alter bone metabolism were excluded, and all other confounding variables that may influence the outcome of endodontic treatment (age, sex, diabetes, smoking and cardiovascular diseases, periapical diagnosis, follow-up, primary or secondary root canal treatment, and tooth type) were considered in the multivariate logistic regression analysis. The patients were treated using different types of statins (simvastatin, atorvastatin, pravastatin, rosuvastatin, lovastatin) at different dosages [10, 20, 40, 80 mg daily], whereas the controls were thirty healthy subjects without AP. All patients meeting the inclusion criteria of the study had completed a follow-up examination 2–5 years post-treatment, including radiographic and clinical evaluation. AP healing was assessed using the periapical index (PAI) [46], which was attributed to the radiographs by two calibrated and expert endodontists [18].

Among the other five articles selected, one was an in vivo animal study [26], and all the others were combined in vitro/in vivo animal studies [22‐25]; however, in respect of the inclusion criteria, only the in vivo experiments part of the research were considered. The animal species employed included Sprague–Dawley rats [22‐25] and Wistar rats [26]. Four studies used a case sample of 10 rats compared to 10 controls [22‐25], and the fifth study analyzed 25 rats: 12 cases, 12 controls, and 1 negative control (without any intervention) [26]. In all experiments, one mandibular molar from each rat was selected [22‐26]. In three studies, AP was induced by accessing the pulp chamber and leaving it open in all treatments time [22‐24]. Alternatively, AP was induced by extirpating the pulp, contaminating the canals with saliva, filling the access with temporary cement [26], or sealing the exposed pulp chambers with amalgam [25]. In all the included studies, the drug compound examined was simvastatin, which was administered to the animals by injections given either on days 1 and 7 [22, 23], at day 0 and then every 5 days until day 21 [24], or from day 20 every 5 days until day 35 [25], depending on the protocol. In one research, the rats were fed using gavage for 20 days, starting 10 days before pulp exposure [26]. The dose of simvastatin administered was 20 mg/kg in all the five animal studies, representing the equivalent of a human dose of about 1.6 mg/kg, which is a little higher than that used in a standard therapy for humans (0.1–1.0 mg/kg per day). Four studies used both immunohistochemistry and imaging, bidimensional radiographs [22‐25], and computerized tomography (CT) [25] to investigate AP, whereas in one experiment, only immunohistochemical analysis was conducted [26].

In all these studies, the protective activity of statins against bone resorption resulting from the inflammatory processes of AP was evaluated. All the experiments included a study group (animals under the simvastatin effect) and a control group (animals receiving placebo). Osteoblasts are the most commonly considered cells. Lai et al. [23] and Yang et al. [25], examined the importance of the balance of apoptosis/autophagy/mitophagy mechanisms of osteoblasts during the development of AP. Both studies evaluated the occurrence of apoptosis through a procedure called the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) reaction, which allows the detection of DNA strand breaks, which are the result of endonuclease activity during the late stage of the apoptotic cascade [23, 25]. Lai et al., also considered the number of osteoblasts with Beclin-1 + , a marker of the autophagic process [23], whereas Yang et al. analyzed hypoxia-induced mitophagy in osteoblasts in the presence of PTEN-induced kinase 1 (PINK-1) in these cells in AP [25]. Shadmehr et al. focused on the expression of osteoprotegerin (OPG) and receptor activator of NF-kappa B LIGAND (RANK-L), fundamental for the regulation of bone apposition in AP, by means of RNA extraction and quantitative real-time PCR [26]. Finally, in two studies [22, 24] the recruitment of histiocytes/macrophages in the resorption lacunae of AP was evaluated by testing the presence of the inflammatory process Cluster of Differentiation 68 (CD68). Furthermore, they analyzed the expression of some inflammatory markers in osteoblasts, such as cysteine-rich angiogenic inducer 61 (Cyr 61), a TNFα-stimulated protein that promotes cell adhesion, chemotaxis, angiogenesis, and regulates tumor growth [22, 24], the chemokine C–C motif ligand 2 (CCL2), which is involved in the recruitment of monocytes, memory T cells, and dendritic cells to the sites of inflammation, and PHOSPHO-Forkhead box class O 3a (p-FoxO3a), an inactivated transcriptional repressor of Cyr61 by phosphorylation [24].

The treatment groups and experimental design employed in each of the included studies are described in detail in Table 1.

The five animal studies evaluated using the ARRIVE guidelines showed a moderate grade of quality [22‐26] (Fig. 2). The cohort study was evaluated using the “Newcastle Ottawa Quality Assessment Form for Cohort Studies” guidelines and exhibited a moderate grade of quality [18] (Fig. 3).

Five animal studies evaluated using the SYRCLE tool presented a moderate risk of bias [22‐26] (Fig. 4).

The cohort study was evaluated with the “Tool to assess risk of bias in cohort studies of CLARITY Group,” and it also showed a moderate risk of bias [18] (Fig. 5).

The eligible studies showed a moderate overall quality and a moderate risk of bias. Considering the scarcity of studies investigating this topic, all studies meeting the inclusion criteria were included in this review to provide the best and most comprehensive available evidence.

All included studies showed a beneficial effect of statins on AP in humans and experimental animal models (Table 1) [18, 22‐26].

The hypothesis that statins could be associated with an increased healing rate of AP was supported by the retrospective cohort study in humans [18]. The rate of healing of AP in 30 patients undergoing treatment with statins, at 2 to 5 years follow-up was significantly higher in the study group (93%) compared to that in the control (70%), even though the cases were older in age than the controls (63 ± 9.7 vs 53.8 ± 16.1 years), and the patients were prescribed different types and dosages of statins. It is important to underline that this altered match between cases and controls, which make the results even more significant, can also be considered the main limitation of this included study.

In animal research, simvastatin appeared to positively affect the development of induced AP. In most studies [22‐26], the radiographic [22‐24], tomographic [25], and image analyses [22‐25] showed that bone resorption was significantly attenuated in animals which were administered subcutaneous injections of simvastatin, either immediately before [22, 23], at the same time [24], or after [25] the induction of AP. This effect was observed in the average sizes [22‐24] and volumes [25] of the AP lesions, which were significantly smaller in the animals treated with statins than in the controls.

Better preservation of bone integrity, manifested as smoother bony contours in the periradicular lesions of the involved teeth, was also observed in the research group as a possible result of a less aggressive osteolytic activity occurring in the lesions [23, 24]. Furthermore, when the most important factors involved in osteoclast differentiation were analyzed [26], the expression of RANKL decreased, while OPG, an inhibitor of the resorptive effects of RANKL, increased in a time-dependent manner in the simvastatin group [26]. Lastly, immunohistochemistry showed that mitophagy (PINK-1) [25], apoptosis (TUNEL) [23, 25], and pro-inflammatory markers (Cyr-61, CCL2, p-FoxO3a, and CD68) [22, 24] were significantly lower in the statin-treated rats. The only autophagy marker considered (Beclin-1) was prominent in osteoblasts and fibroblasts of the study group, suggesting that autophagy exerts a protective role against cell death during the development of AP [23]. These data demonstrate that there is an association between statin administration and protective action during the inception of AP in animal models.