Gingival enlargement (GE) stands as a common complication in patients undergoing fixed orthodontic appliance therapy.^[1] Although the etiology of GE has been explored in the literature, the precise pathophysiological mechanisms underlying GE induced by orthodontic treatment remain largely uncharted. Some studies suggest that both chemical and mechanical irritation, arising from dental cements and orthodontic equipment, may contribute to GE.^[2] It has also been postulated that GE can create pseudo-pockets, providing an environment conducive to the colonization of anaerobic microbiota.^[3] Moreover, the host’s response to microbial challenges mediated by inflammatory cytokines such as interleukin-1β (IL-1β) and transforming growth factor-β1 can lead to increased production of amorphous ground substance, culminating in GE.^[1,4] Furthermore, there is evidence to suggest that even the gradual release of nickel near orthodontic appliances at low levels can initiate epithelial proliferation, ultimately resulting in gingival overgrowth.^[5] Orthodontic patients are often assumed to exhibit compromised periodontal health.^[6] However, in contrast, some studies indicate that periodontal conditions do not necessarily worsen during orthodontic treatment, even in the presence of plaque-retentive devices.^[7] There is a debate as to whether orthodontic therapy is detrimental or beneficial to the long-term status of periodontal structures.^[8]

Orthodontic appliances create conditions favorable for dental plaque accumulation and pose challenges for maintaining adequate oral hygiene, particularly in interdental areas that require adjunct cleansing aids. Consequently, ensuring an optimal level of plaque control may require additional time, skills, and effort, and it is not always achieved successfully. In addition, orthodontic patients experience alterations in the prevalence of periodontal pathogens, specific to both local and site-specific contexts.^[9]

Notably, GE can occur in orthodontic patients even when oral hygiene is acceptable and there are no clinical signs of inflammation.^[10] This phenomenon can be attributed to the gingiva’s response to the mechanical forces exerted by orthodontic appliances, resulting in the remodeling of the periodontium and tooth movement. Consequently, the inflammation observed in hypertrophied gingiva may be considered an additional event, especially when access to the tooth surface is already limited in these situations.

During orthodontic treatment, the remodeling process involves several cytokines and enzymes, with matrix metalloproteinases (MMPs) playing a prominent role. Among these MMPs, MMP-2 (gelatinase A) and MMP-9 (gelatinase B) are members of a zinc- and calcium-dependent endopeptidase family, also known as human gingival gelatinases or Type IV collagenases. Fibroblasts, endothelial cells, and osteoblasts express MMP-2, while keratinocytes, polymorphonuclear leukocytes, and macrophages synthesize MMP-9.^[11,12] Previous research has delved into their roles in collagen turnover, inflammation initiation, wound healing, the development of periodontal disease, and periodontal remodeling during orthodontic therapy.^[12,13] MMPs play a crucial role in the physiological remodeling of the periodontal ligament and the tissue’s response to mechanical stressors induced by orthodontic treatment.^[14] Dysregulation of MMP activity has been implicated in pathological processes, particularly chronic inflammation in periodontal diseases.^[15,16] MMPs are typically expressed in low quantities in their latent form and are subsequently activated through proteolytic or oxidative/nitrosative mechanisms. Unlike enzyme-linked immunosorbent assay (ELISA), which can quantitatively measure protein levels, they cannot assess enzyme activity.^[17]

Although Şurlin et al. conducted studies exploring the potential association between MMP concentration and GE, none of these studies considered the enzymes’ degree of activity and functional capacity as potential influencing factors in GE.^[10,15] To our knowledge, no studies have assessed the activity of MMPs in orthodontic treatment-induced GE. Therefore, this study aims to investigate the potential relationship between the activity of MMP-2 and MMP-9 in orthodontic patients with GE.

A total of 50 subjects participated in our study, divided into two groups: those with GE (case group) and those without GE (control group). The sociodemographic characteristics of the study sample are summarized in Table 1, showing no significant differences between the case and control groups regarding gender and age.

Table 2 presents the results of the clinical examinations, where T1 represents the 4^th month after the initiation of orthodontic treatment and T2 represents the 5^th month. This notation is consistent throughout all tables and figures.

In Figure 1, gelatin zymograms depicting MMP2 and MMP9 gelatinolytic activity in subjects with and without GE at T1 and T2 are presented. As shown in Figure 2, the activity of MMP-9 was more pronounced in the case group compared to the control group. However, this difference reached statistical significance only at T1 (the 4^th month of orthodontic treatment), with P = 0.004. In T2 (the 5^th month), the difference did not reach a statistically significant level, with P = 0.132.

Table 3 presents the distribution of the case group according to gingival enlargement score categories. Table 4 illustrates that MMP-9 activity increased in both the case and control groups from the 4^th to 5^th months of treatment. However, this change was not statistically significant in either of the groups, with P > 0.05.

Furthermore, no significant correlations were found between MMP-9 activity and the GE score at T1 and T2, with P = 0.328 and P = 0.224, respectively.

The conventional understanding of GE in orthodontic patients attributes it to inflammation resulting from poor dental hygiene and plaque accumulation. However, there is a growing body of research suggesting that mechanical stress may also play a role in gingival erosion and periodontal remodeling during orthodontic treatment.^[10] MMPs, which are enzymes involved in tissue remodeling during tooth movement,^[14,21] are potential candidates for influencing GE in individuals undergoing fixed orthodontic therapy.^[10,15] The primary objective of our study was to investigate whether the activity of MMP-2 or MMP-9 undergoes changes in orthodontic patients with GE.

Previous studies have compared the levels of different MMPs in various periodontal health conditions using techniques such as ELISA, zymography, and Western immunoblotting.^[22,23] Maeso et al. proposed that imbalanced levels of MMPs and tissue inhibitors of metalloproteinases (TIMPs) in gingival crevicular fluid (GCF) might be responsible for initiating tissue breakdown in periodontitis. They employed ELISA to measure concentrations of MMP-2, MMP-9, and TIMP1, finding a significant decrease in TIMP1, a modest increase in MMP-9, and a nonsignificant decrease in MMP-2 in periodontal disorders.^[22] Similarly, Soell et al. argued that MMP-induced tissue destruction in periodontal disease is related to the imbalance between increased levels of enzymes and their inhibitors rather than the activation of proenzymes. They reported a substantial increase in MMP-2 levels when comparing GCF samples from periodontal patients to those from healthy individuals.^[24] The roles of MMP-2 and MMP-9 in periodontal tissue degradation have been emphasized, and their activity levels have been shown to significantly decrease after effective periodontal treatment.^[12] Séguier et al. indicated active forms of MMP-9 as a good indicator of tissue breakdown and clinical severity of periodontal disease. ProMMP-9 and active MMP-2, however, did not substantially rise. Their findings showed a slight but marginal reduction in pro MMP-2.^[23] Some research shows that latent MMP-2 and MMP-9 are found in both chronic periodontitis and healthy periodontium, while active MMP-2 is exclusively present in cases of periodontitis.^[25]

However, there are limited studies investigating the possible role of MMPs and their activity in GE. Şurlin et al. employed a double immunofluorescence technique on enlarged gingival tissues and found a higher expression of MMP-9 and type IV collagen in individuals with GE.^[15] Despite the high expression of MMP-9 in the expanded gingival tissues, they concluded that the increased quantity of MMP-9 is insufficient to damage the basal membrane. Their hypothesis was that mechanical stress, rather than the inflammatory process, is the primary reason for the elevated MMP-9 levels in the GCF of orthodontic patients and the onset of GE.^[15,26] However, the enzyme used in their immunohistochemical examination targeted a sequence of amino acids present in both active and latent forms of MMP-9 and could not differentiate between the proenzyme and active form of MMP-9.^[15] The same authors observed elevated levels of MMP8 in GCF and hypothesized that MMP8 levels in GCF could serve as an indicator of GE onset. They found that MMP8 peaks 4–8 h after the initiation of orthodontic treatment, decreases in patients with normal gingiva, and continues to rise in certain individuals until their gingiva becomes enlarged. Immunohistological examination of excised tissue from gingivectomy procedures revealed higher expression of MMP8 in the overgrown gingiva of orthodontic patients. Thus, they postulated that the amount of MMP8 in GCF could be an indicator of GE onset.^[10]

In our study, we opted to monitor the activity of gelatinases as an indicator of GE rather than their quantity, as the total amount of MMPs often includes latent or inactive enzyme forms. We employed zymography, which involves the electrophoretic separation of proteins using a polyacrylamide gel containing a proteolytic substrate. Following denaturing (but nonreducing) electrophoresis, the proteins were renatured and subjected to an appropriate solution for proteolytic activity. Clear zones of lysis on the stained gel indicated active proteinases, allowing us to distinguish between active and latent forms of MMPs based on their molecular weight and their ability to lyse the gelatin substrate, commonly used for MMP-2 and MMP-9.^[27] Substrate zymography is a practical method for assessing the activity of MMP isoenzymes.^[17]

Despite the structural similarities of MMP-2 and MMP-9 and their substrate selectivity, they are regulated by distinct mechanisms. For instance, the basal level of MMP-9 is typically low, and its secretion from inflammatory cells is induced by cytokines such as tumor necrosis factor-alpha (TNF-α), while MMP-2 is produced by various cell types and its secretion does not usually require cytokine induction. Furthermore, MMP-9 plays a pro-inflammatory role through the cleavage of IL-8, significantly amplifying neutrophil chemoattraction^[28] and converting proTNF-α and proIL-1β into their active pro-inflammatory forms.^[29,30] In contrast, MMP-2 acts to inhibit inflammation^[31] and has been shown to degrade inflammatory mediators such as monocyte chemoattractant protein-3.^[32,33]

Our study revealed a noticeable increase in salivary MMP-9 activity in subjects with GE compared to those without GE at T1. However, the intergroup difference diminished to a statistically insignificant level after 1 month (T2). In addition, our results did not demonstrate a correlation between MMP-9 enzyme activity and the GE score. Our findings align with those of Kim et al., who analyzed the expression levels of MMP-2 and MMP-9 in gingival tissues of periodontitis patients using Western blot analysis and assessed their enzymatic activity via gelatin zymography. However, after 1 month, the intergroup difference dropped to a statistically insignificant level. In addition, our results did not indicate a link between MMP-9 enzyme activity and GE score. In our investigation, the activity of MMP-9 differed between the case and control groups, corroborating the results of Kim et al. They analyzed the expression level of MMP-2 and MMP-9 in gingival tissues of periodontitis patients using Western blot analysis and their enzymatic activity by gelatin zymography.^[16] They concluded that the expression of MMP-9 was positively correlated with its activity; suggesting that MMP-9 amount and activity can be a predictive biomarker for the progression of periodontitis. However, this study did not observe any correlation between the expression level and activity of MMP-2. Similarly, our test on GE did not detect any activity of MMP-2 in saliva of patients with GE. In our study, the MMP-9 activity increased from the 4^th to 5^th months after the beginning of orthodontic treatment in subjects with GE, while there was not any correlation between enzyme activity and GE score. Kim et al. reported a positive correlation between the expression level and activity of MMP-9 and suggested that MMP-9 quantity and activity could serve as predictive biomarkers for the progression of periodontitis. However, they did not observe any correlation between the expression level and activity of MMP-2.

In our study, we did not detect any activity of MMP-2 in the saliva of patients with GE. Moreover, MMP-9 activity increased from the 4^th to 5^th months after the initiation of orthodontic treatment in subjects with GE. Nevertheless, no correlation was found between enzyme activity and the GE score.

One limitation of our study was our inability to quantify the enzyme quantity alongside assessing its activity. While measurements of MMP levels and activity might have been used to precisely determine the involvement of MMPs in GE, investigating the activity of enzyme inhibitors such as TIMP1 could provide further insight into the underlying mechanisms.

Furthermore, our data revealed a significant inter-individual heterogeneity. Factors such as variations in dental biofilm composition, its accumulation pattern, and differences in bacterial flora may have interfered with our measurements. In addition, we measured the target mediators in saliva, but site-specific monitoring of MMPs in the GCF of teeth exhibiting GE could have yielded different results.

In summary, this study contributes to our understanding of the role of MMPs, specifically MMP-9, in the pathogenesis of GE in orthodontic patients. While we observed significant differences in MMP-9 activity between individuals with and without GE at the 4^th month of treatment, the exact mechanisms and factors involved require further investigation. The complex interplay between mechanical stress, inflammation, and MMP activity in the context of GE warrants continued exploration and a more in-depth understanding of this phenomenon.

Our study provides insights into the potential role of salivary MMP-2 and MMP-9 activity in orthodontic-induced GE. The study revealed that while MMP-2 activity was undetectable, MMP-9 activity increased during the 4^th month of orthodontic treatment. However, this increase in MMP-9 activity did not exhibit a significant correlation with the degree of GE. These findings suggest that MMP-9 activity may play a role in the development of GE among orthodontic patients. Further research is needed to unravel the precise mechanisms underlying this phenomenon and its clinical implications.