**3. Discussion**

The prevalence of GOIA might be as high as 38%, and it is 3.3 times more common in men than in women [20,21]. The pathogenesis may be different for different drugs, even if the oral manifestations of gingival overgrowth are similar. GOIA starts as an enlargement of the interdental papilla of keratinized portions of the gingiva, and it is characterized by an increase in the connective tissue component. Bacteria accumulation appears to be an important uncomfortable effect of GOIA. GOIA may impair oral hygiene and lead to increased oral infections. Oral infection itself is a cause of gingival overgrowth [22]. In addiction, oral infection can potentially impair systemic health and could possibly compromise the general health of patients [22].

The mechanism of action of GOIA is still unknown, however it may be a consequence of the interaction between gingival fibroblasts, cellular and biochemical mediators of inflammation, and drug metabolites [22,23].

Gingival overgrowth is documented more frequently after intake of with phenytoin and rarely with others antihypertensive [24]. Furthermore, poor oral hygiene is indicated as an important risk factor for the expression of GOIA [25,26].

Cross-sectional studies have reported the relationship between bacterial plaque and GOIA. In fact, as previously reported, GOIA can favour the accumulation of bacterial plaque, which in turn determines gingival inflammation, causing gingival overgrowth [26]. The underlying mechanism of GOIA still remains to be fully understood, however two main inflammatory and non-inflammatory pathways have already been suggested [27–29]. One hypothesis may be that amlodipine may induce the alteration of collagenase activity as a consequence of decreased uptake of folic acid, blockage of aldosterone synthesis in adrenal cortex, and consequent feedback increase in the adrenocorticotropic hormone level and the up-regulation of keratinocyte growth factor [30]. Besides, inflammation may be a consequence of the toxic effect of amlodipine in periodontal pocket associated with C pathogens, leading to the up-regulation of several cytokine factors, such as transforming growth factor-beta 1 (TGF-β1) [9]. Another pathogenic mechanism of GOIA is focusing on the effects of amlodipine on gingival fibroblast metabolism and genetic predisposition. In fact, only a subgroup of patients that were treated with this amlodipine will develop GOIA, so it has been hypothesized that these individuals show an abnormal susceptibility to the drug. In fact, elevated levels of protein synthesis, most of which is collagen, characterize the fibroblast of GOIA in these patients. Treatment of GOIA is generally targeted on drug substitution and preventive protocols [12,31]. Surgical intervention is recommended when these measures fail to cause the resolution of GOIA. These treatment modalities, although effective, do not necessarily prevent the recurrence of the lesions [12]. Surgery for treatment of GOIA must be carefully assessed and it is normally performed for cosmetic/aesthetic needs before any functional consequences are present [1,32]. Most reports of GOIA have required surgical intervention [25].

To our knowledge, our study is the first one analysing the effect of amlodipine on genes that belong to the "Extracellular Matrix and Adhesion Molecules" pathway. In this study, gingival fibroblasts were treated for 24 h with 1000 ng/mL of amlodipine. The gene expression profile of 12 genes that belong to the "Extracellular Matrix and Adhesion Molecules" pathway was analysed. Most of the significant genes were up-regulated. (*CTNND2*, *COL4A1*, *ITGA2*, *ITGA7*, *MMP10*, *MMP11*, *MMP12*, *MMP26*), except for *COL7A1*, *LAMB1*, *MMP8*, and *MMP16*, which were down-regulated. These proteins preferentially induce extra cellular matrix deposition. This study demonstrated that, in human gingival fibroblasts that were cultivated in vitro, amlodipine could promote the activities of genes belonging to the "fibroblast matrix and receptors".

It might be part of the underlying reason for the persistent overgrowth of gingiva that was seen when bacterial plaque and local inflammation are present during amlodipine therapy. In fact, GOIA does not allow patient to maintain a good oral hygiene, and this is the reason why GOIA always determines the presence of bacterial plaque and inflammation, which in turn determines gingival overgrowth.

The data presented here sugges<sup>t</sup> that amlodipine may contribute to an extracellular matrix deposition of human gingival fibroblasts inducing gingival overgrowth.

### **4. Materials and Methods**

### *4.1. Primary Human Fibroblast Cells Culture*

We used cells from ATCC® Cell Lines. The cryopreserved cells at the second passage were cultured in 75 cm<sup>2</sup> culture flasks containing DMEM medium (Sigma Aldrich, Inc., St Louis, Mo, USA) supplemented with 20% fetal calf serum, antibiotics (Penicillin 100U/ml and Streptomycin 100 micrograms/ml-Sigma Aldrich, Inc., St Louis, Mo, USA).

Cell cultures were replicated for subsequent experiments and maintained in a water saturated atmosphere at 37 ◦C and 5% CO2.

### *4.2. Cell Viability Test*

A stock solution of amlodipine 1 mg/mL was prepared. Further dilutions were made with the culture medium to the desired concentrations just before use. The cell lines were seeded into 96-well plates at a density of 104 cells per well containing 100 μL of cell culture medium and incubated for 24 h to allow cell adherence. Serial dilution of amlodipine (5000 ng/mL, 2000 ng/mL, 1000 ng/mL, 500 ng/mL, 100 ng/mL) was added (three wells for each concentration). The cell culture medium alone was used negative control.

After 24 h of incubation, cell viability was measured while using PrestoBlue™ Reagent Protocol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Briefly, the PrestoBlue™ solution (10 μL) was added into each well containing 90 μL of treatment solution. The plates were then placed back into the incubator for 1 h, after which absorbance was measured at wavelengths of 570 nm excitation and 620 nm emission by an automated microplate reader (Sunrise™, Tecan Trading AG, Switzerland). Comparing the average absorbance in drug treated wells with average absorbance in control wells exposed to vehicle alone determined the percentage of viable cells.
