## 4 Discussion  
Gingival enlargement occurs as an adverse reaction in patients treated with calcium channel blocker agents (CCBs). It has been reported that DIGO is the most frequent periodontal side effect of CCBs, leading to mastication and speech problems, altering aesthetics, complicating oral hygiene maneuvers, and increasing the risk of bacterial infection [27]. CCBs take action in calcium metabolism: these drugs can decrease cellular Ca\({}^{2+}\) influx and consequently reduce the uptake of folic acid, leading to a decreased synthesis of active collagenase, implicated in collagen degradation. Collagen degradation inhibition results in collagen accumulation [28; 29]. In our study, fibroblasts were treated with nifedipine solution at a concentration of 1000 ng/mL for 24 h, and the obtained data showed a significant downregulation of genes coding for metalloproteinase (MMP) 11, 16, and 26. MMPs are enzymes involved in the connective tissue catabolic turnover, whose activation inhibition leads to connective tissue accumulation [30; 31]. These results confirm that the increase of connective tissue collagen fibers induced by CCBs derives from the inhibition of collagen extracellular degradation pathways rather than from the increase in its production [21]. Downregulation was also observed for integrins ITGB4 and ITGA6, transmembrane cell-matrix adhesion receptors in fibroblasts that are responsible for collagen phagocytosis (collagen intracellular degradation pathway). The inhibition of MMPs expression occurs not only in nifedipine-induced GO, but it is also observable in gingival enlargement associated with immunosuppressive administration. The research of [PERSON] et al. [32] recorded a downregulation of MMP8, MMP11, MMP15, MMP16, and MMP24 in gingival fibroblasts treated with cyclosporine and mycophenolate mophetil solutions. Many other authors demonstrated that anticonvulsant and immunosuppressive drugs are able to reduce the expression of \(\alpha\) and \(\beta\) integrins or to interfere with collagen adhesion, thanks to pro-inflammatory cytokines action and, consequently, altering collagen phagocytosis mechanisms [33; 34; 35]. [PERSON] et al. demonstrated, in particular, that the lack of collagen degradation, in a sample of 20-day-old rats treated with nifedipine, was associated with the storage of Type I collagen [36]. According to [PERSON] et al. [37], nifedipine-induced gingival enlargement is characterized by an increase in Transforming  
Figure 2: Gene expression profile of fibroblasts treated with nifedipine. growth factor -\(\beta\) signaling, known as a mediator of fibrotic processes and able to regulate extracellular matrix remodeling and inflammatory response [36; 38]. The authors of this study demonstrated that TGF-\(\beta\), in nifedipine-induced GO, causes an upregulation of gingival fibroblasts periostin, a matricellular protein involved in functional and structural regulation of connective tissue [39]. TGF-\(\beta\) also stimulates fibrobl population and ECM deposit of fibronectin and glycosaminoglycans [40]. According to the literature, interleukin 1\(\beta\) (IL-1\(\beta\)) and IL-6 have been demonstrated to play a pathogenic role in drug-induced gingival fibrosis [12; 41]. The authors of our study also assessed CDH1 (E-cadherin) level by performing an enzyme-linked immunoassay (ELISA): an increase of its levels was recorded in treated fibroblasts vs. untreated control, confirming the gene expression results obtained by real-time PCR. Cadherins 1 are calcium-dependent cell adhesion proteins and are involved in mechanisms regulating cell-cell adhesion, mobility, and proliferation of epithelial cells [42]. This data is in contrast with those provided by [PERSON] et al. [43], who studied the effect of phenytoin, cyclosporine, and nifedipine administration on gingival tissues' basement membranes. [PERSON] et al.