Apical periodontitis is a common inflammatory disease that leads to tooth loss along with caries. Apical periodontitis represents a local immune response against invading microorganisms from an infected root canal space to the periapical region.^[1] Despite advances in imaging, diagnosing pulp and periapical issues remains difficult, therefore exploring molecular diagnostics may improve understanding and enable the development of chairside tests. However, limitations persist due to the inability of current dental imaging methods to assess soft-tissue inflammation or bacterial infiltration in the dental pulp.^[2]

Host cells release various inflammatory mediators, proinflammatory cytokines, and growth factors as an immune response during periapical infection.^[3] Matrix metalloproteinases (MMPs) are vital endopeptidases that exhibit significant importance.^[4] The presence of pathogens stimulates host cells to increase the release of MMPs, which are considered indirect mechanisms in tissue destruction during periodontitis. The healthy periodontal ligament is protected from the proteolytic attack of MMPs by Tissue Inhibitors of Metalloproteinases (TIMPs). In chronic periodontitis, the level of TIMP is low and insufficient to inhibit MMPs. As a result, progelatinase (pro-MMP-9) is activated and it leads to the accumulation and activation of inflammatory cells such as lymphocytes and neutrophils. Consequently, changes in inflammatory mediators and secretion of inflammatory proteases will occur.^[5]

MMP-9 or gelatinase B is reported to be a multidomain enzyme that functions in both acute and chronic inflammatory diseases.^[6] MMP-9 is a gelatinase that is mainly secreted by neutrophils, and during the active and progressive phase of periodontitis, the level of MMP-9 in the periodontal crevicular fluid increases significantly.^[7] MMP-9, by breaking down type IV collagen, disrupts basement membranes associated with tumor infiltration and metastasis.^[8] It is also necessary to initiate clastic bone resorption by removing collagen from the bone structure before demineralization begins. MMP-9 plays a crucial role in periodontal bone destruction by degrading extracellular matrix components and promoting inflammatory responses, which accelerates tissue breakdown and bone loss in periodontal disease.^[7,9]

In addition, it has been suggested that root canal treatment (RCT) can interfere with the expression of MMPs.^[10] Therefore, MMP-9 can be used as a diagnostic biomarker for the accurate and timely diagnosis of the periapical condition.^[2] This study aims to compare the level of MMP-9 in gingival crevicular fluid (GCF) in the conditions of acute and chronic apical periodontitis before and after RCT in comparison with the healthy periapical tissue. The null hypothesis was that there is a difference in MMP-9 levels in the GCF between cases of acute and chronic periodontitis, between periodontitis and healthy cases, and before and after RCT.

The statistical analysis indicates that MMP-9 values before and after RCT in the study groups had a normal distribution. Pairwise comparisons for MMP-9 in the studied groups showed no notable difference between the MMP-9 values before treatment of the acute group (0.48 μg/μL) and the normal group (0.476 μg/μL) (P = 1) [Table 1]. However, there was a significant difference between the normal and chronic (0.534 μg/μL) groups before treatment (P = 0.0001). A noticeable difference was found between acute and chronic groups both before and after the RCT (P = 0.001). The MMP-9 levels after the treatment for the acute and chronic group were 0.445 and 0.491 μg/μL, respectively. There was no significant difference between the normal group values and any of the two groups after the treatment (P = 0.009).

The results of the paired t-test indicate that there were remarkable differences between the conditions before treatment and after treatment. In the acute group, the average MMP-9 after RCT is 0.035 μg/μL less than before treatment, while for the chronic group, this difference was 0.043 μg/μL [Figure 1].

For chronic conditions, the correlation between gender and MMP-9 levels before and after treatment was − 0.076 and 0.048, respectively. These results suggest weak correlations. For acute conditions, the correlation between gender and MMP-9 levels before and after treatment was − 0.206 (moderately negative) and 0.202 (moderately positive), respectively [Figure 2]. For the healthy group, the correlation is moderately positive (0.207), indicating a moderate positive, but not strong relationship between gender and MMP-9 levels in healthy individuals.

Furthermore, regarding the correlation between age and MMP-9 levels, for chronic conditions, before and after treatment, it was 0.104 and 0.159, respectively. For the acute group, it was −0.027 and −0.256 before and after RCT, respectively. The strongest correlation is in the healthy group (0.39), suggesting as age rises, the MMP-9 level increases in this group [Figure 3].

In this study, we evaluated the level of MMP-9 in the GCF before and after RCT in teeth with acute apical periodontitis, chronic apical periodontitis, and in healthy teeth. The results indicate that the local levels of MMP-9 increased in the two experimental groups with pulp and periapical inflammation and decreased following RCT.

Various studies investigated the levels of MMP-9 to track the changes in pulp and periapical inflammation status.^[11] For instance, Ballal et al. reported that the up-regulation of MMP9 in pulp wound has been known as a signature element for the gene expression in irreversible pulpitis.^[12] Moreover, MMP-8 is suggested as one of the best diagnostic biomarkers for periodontal diseases and showed a high specificity and sensitivity on disease severity in a point-of-care mouthwash assessment.^[13] Furthermore, Tsai et al. showed that the expression of MMP-9 mRNA in the pulp of teeth with inflammation increased compared to healthy teeth.^[14]

Under normal conditions, the healthy periodontal ligament is protected from the proteolytic attack of MMPs by TIMPs. In chronic periodontitis, the level of TIMP is low and therefore, insufficient to inhibit MMPs. In this study, before the treatment, there was no significant difference between the MMP-9 level of the normal group and the acute periodontitis group. In contrast, there was a notable difference between the normal and chronic periodontitis groups. This can be due to the nature of the inflammatory response in acute versus chronic conditions. Acute apical periodontitis involves a rapid and transient inflammatory response, where MMP-9 levels may not have had sufficient time to rise significantly above those in healthy teeth.^[15] In contrast, chronic apical periodontitis is characterized by prolonged and sustained inflammation, leading to consistently higher MMP-9 levels due to ongoing tissue remodeling and destruction.^[16]

Regarding the changes in MMP-9 level after RCT, similar to our study, Shin et al. reported that the level of MMP-8 and substance P were measured in GCF before, during, and after RCT, and a decrease in the amount of inflammatory cytokines was observed after the treatment as a result of the reduction of inflammation.^[17] Furthermore, Al-Abdulla et al. evaluated the serum level of MMP2, 2 years following nonsurgical root canal re-treatment and peri-apical surgery, and found that it can be a potential biomarker of inflammation indicating the successful treatment of chronic apical periodontitis.^[18] In addition, Akbal Dincer et al. compared neurokinin A, substance P, interleukin-8, and MMP-8 levels in healthy and symptomatic irreversible pulpitis, finding significant increases in these biomarkers in GCF of symptomatic teeth using ELISA technique, with decreases after pulp removal.^[19]

In contrast to our study, Ahmed et al. reported that after the RCT procedure, the amount of MMP-9 in symptomatic periapical periodontitis lesion sections was considerably higher than in asymptomatic periodontitis; however, this amount did not differ notably in acute and chronic groups after the treatment in our study.^[1] Moreover, Gusman et al. concluded that the level of MMP-1, MMP-2, and MMP-3 in acute pulpitis was higher compared to the healthy opposite tooth of the same person.^[20] In addition, Victor et al. reported that the amount of MMP-9 in smokers with chronic periodontitis is higher than that of nonsmokers with chronic periodontitis.^[5]

In contrast to our study, in the study of Zehnder et al., it was shown that the amount of MMP-9 was detectable only in teeth with symptomatic pulpitis and could not be measured in healthy teeth.^[6] This can be due to the different sampling methods and measuring the dentinal fluid instead of GCF. Different sources have been used for sampling local biomarkers such as apical lesions, periodontal ligament, gingival fluid, dentinal fluid, pulp tissue of extracted teeth, periapical exudate, and saliva.^[1,5] Pattamapun et al. assessed MMP-2 levels in root-canal exudates during RCT and it notably decreased in teeth with necrosis after treatment.^[21] Getting samples from root canal exudates can be highly influenced by instrumentation and chemical debridement that leads to the removal of residual microorganisms. Ahmed et al. figured out that, the possibility of measuring MMP in the dentinal fluid is low.^[1] Also regarding getting samples from apical lesions, periapical surgery to remove periapical granuloma and tooth extraction to remove pulp tissue for this purpose is not ethically acceptable and may not be accepted by patients. Therefore, GCF was chosen as our study sample since it is a simple and noninvasive method and it is more frequently used in studies.

In our study, each canal was irrigated with 2 mL of 2.5% sodium hypochlorite (NaOCl, Cerkamed, Poland), and a final irrigation with 5 mL of distilled water (Farazdental, Iran) was performed. Higher concentrations of NaOCl (e.g., 5% and 5.25%) have been shown to further decrease MMP activity. Previous studies have demonstrated that while NaOCl and chlorhexidine reduce MMP expression in radicular dentin, EDTA can increase MMP levels.^[22,23] Therefore, the use of 2.5% NaOCl aligns with these findings. By adhering to these standardized procedures, we aimed to minimize variability and focus on the specific outcomes related to the conditions under investigation.

To collect samples, different tools can be used such as PVDF membrane (polyvinyl fluoride filter membrane), medium size paper cone, and filter paper strip (standard filter paper tape).^[2,24] The present study used a medium-sized paper cone due to its availability and easier use. Similar to Shin et al. study, a number of paper cones were placed in the tooth sulcus for 30 s and then placed in Tris HCL pH: 7.5 buffer solution before transferring to the laboratory.^[17]

In order to analyze the MMP-9 of the samples, several laboratory analyses such as Zymography, Densitometry, ELISA, and Immuno Western Blot can be used.^[10,25] In this study, due to the high accuracy of the ELISA, this test was used for laboratory tests.^[26] Avellan et al. measured the molecular forms of MMP-8 in the GCF of stimulated and nonstimulated teeth by Western immunoblot, and MMP-8 levels by quantitative immunofluorometric assay and demonstrated elevations in mesenchymal type MMP-8 isoforms in stimulated teeth with pulpal pain.^[24] In contrast with these findings, Kritikou et al. found no significant difference in the level of MMP9 in healthy and symptomatic irreversible pulpitis using the ELISA technique.^[27]

Future research should incorporate different groups using various irrigants, concentrations, obturation techniques, and sealer types to compare their effects on MMP-9 levels. Examining these variables separately could provide deeper insights into how specific clinical procedures influence outcomes. Furthermore, a larger sample size is needed to provide a more precise correlation between the study variables.

Our results indicate that MMP-9 levels in GCF rise during periapical inflammation but decline following endodontic treatment. MMP-9 is a potential diagnostic biomarker for both pulp and periapical inflammation, offering insights into these clinical conditions and guiding future treatment approaches.