The Influence of New Silicate Cement Mineral Trioxide Aggregate (MTA Repair HP) on Metalloproteinase MMP-2 and MMP-9 Expression in Cultured THP-1 Macrophages

The aim of the present study was to investigate the new silicate cement mineral trioxide aggregate (MTA Repair HP) with respect to its effect on the inflammation process involving the tooth and periodontal tissues. The composition of MTA Repair HP was supplemented with plasticizer agents which can have a negative effect on the modulation of tooth inflammation. The silicate-based material in question is widely used in regeneration of the pulp-dentin complex, treatment of perforations of various locations in the tooth, as well as in surgical treatment of the complications of periapical tissue. The improved bioceramic restorative cement can affect the expression of metalloproteinases MMP-2 and MMP-9 in monocytes/macrophages involved in modulation of inflammation and regenerative processes of the tooth and periodontal tissues. The novel aspect of the present study lies in the application of the model of THP-1 monocyte/macrophage and applying the biomaterial in direct contact with the cells. Hence, it provides a representation of clinical conditions with respect to regenerative pulp and periodontal treatment with the use of MTA Repair HP. A lack of macrophage activation (as measured with flow cytometry) was found. Moreover, the study identified a lack of expression stimulation of the studied metalloproteinases (with the use of Western blotting and fluorescent microscopy). Similarly, no increase in MMP-2 and MMP-9 concentration was found (measured by ELISA method) in vitro when incubated with MTA Repair HP. Based on the results it can be concluded that new MTA Repair HP does not increase the inflammatory response in monocytes/macrophages associated with the activity of the described enzymes. It can also be speculated that they do not affect the process of dentin regeneration in which MMP-2 and MMP-9 play significant roles.


1S. MMP-2 and MMP9 activity in macrophages measurement by zymography method
The samples (25µ g) were mixed with Laemmli Sample Buffer (Bio-Rad, Germany) and loaded into wells. The proteins were separated in 7.5% polyacrylamide gels containing 1 mg/ml gelatin and sodium dodecyl sulfate (SDS). After electrophoresis, the gels were washed with renaturing buffer (2.5% Triton™ X-100, Sigma-Aldrich, Germany) and incubated in developing buffer (50 mM Tris, 10 mM CaCl2, 0.02% NaN3, pH=7.4, Sigma-Aldrich, Germany) overnight in 37°C. The gels were washed with water and incubated for 1h in Coomassie Brilliant Blue R-250 (Thermo Fisher Scientific, USA) staining solution. The staining buffer was removed and the gels were incubated with destaining solution (10% methanol, 5% acetic acid, Merck, Germany) until areas of proteolytic activity were clearly visible. Protease bands were detected by absence of Coomassie Brilliant Blue R-250 staining of digested gelatin. Normal serum was used as a control. The bands were qualitatively and quantitatively analyzed with Image Lab Software 6.1.0 (Bio-Rad Laboratories, Inc.).

A.
B. Figure S2. MMP-2 metalloproteinase activity in monocytes (A) equal protein amounts (25 μg) of protein was prepared for gelatin zymography analysis (B) The densitometry values of the proteolytic bands corresponding to MMP-2 is presented as pixel intensity × mm 2 and were normalized to actin to correct for loading. Cells were cultured with mineral trioxide aggregate (MTA Repair HP) for 24 h (MTA24) and 48 h (MTA48) in RPMI medium with 10% FBS. Following incubation, the cells were scraped and protein concentration was measured. The control cells were incubated in 10% FBS RPMI medium for 24h -

kDa MMP-2
control 24 (C24) or for 48h -control 48 (C48). The experiments were conducted as three separate assays. There was no statistically significant differences vs apropriate control group.
Monocytes incubated in the presence of MTA Repair HP for 24 and 48 h (MTA24 vs MTA48) did not show statistically significant changes in activity of the analysed enzyme (p≥0.05, Mann Whitney U test).

A.
B. Figure S3. MMP-2 metalloproteinase activity in macrophages (A) equal protein amounts (25 μg) of protein was prepared for gelatin zymography analysis (B) The densitometry values of the proteolytic bands corresponding to MMP-2 is presented as pixel intensity × mm 2 and were normalized to actin to correct for loading. Cells were cultured with mineral trioxide aggregate (MTA Repair HP) for 24 h (MTA24) and 48 h (MTA48) in RPMI medium with 10% FBS. Following incubation, the cells were scraped and protein concentration was measured. The control cells were incubated in 10% FBS RPMI medium for 24h -control 24 (C24) or for 48h -control 48 (C48). The experiments were conducted as three separate assays. There was no statistically significant differences vs appropriate control group.   There was no statistically significant differences vs appropriate control group.
The results of our study confirm that mineral trioxide aggregate (MTA Repair HP) does not alter MMP-2 and MMP-9 activity in the cultured monocytes/macrophages.

2S. Chemical properties of MTA
Three powder ingredients basically constitute MTA: portland cement (75%), bismuth oxide (20%) and gypsum (5%). The patent specification of MTA states that it contains calcium oxide (50-75 wt %) and silicon oxide (15-20 wt %), which together constitute 70-95% of the cement. When the raw materials are blended, tricalcium silicate, dicalcium silicate, tricalcium aluminate, tetracalciumaluminoferrite are produced [1]. MTA is available in two colours: grey and white MTA, with the major difference being the concentrations of Al2O3, MgO and FeO [24]. The traditional composition of MTA was known to show some disadvantages, such as long setting time, tooth and marginal gingiva discolouration or difficult handling [2,3]. The latter disadvantage was frequently reported when performing filling of root-end cavities and furcation or root perforation [4]. However, to counter the negative aspects of MTA application, new formulations have been introduced recently, such as MTA Repair HP (Angelus, Londrina, PR, Brazil) or MTA Vitalcem [2]. The main components of MTA HP powder are tricalcium silicate, dicalcium silicate, tricalcium aluminate, calcium oxide, calcium carbonate (filler material) and calcium tungstate (radiopacifier). The components of the liquid supplied for mixing with the cement powder are water and plasticizing agent. According to the manufacturer, in comparison with White MTA, the new material shows superior properties such as high-plasticity and improved physical properties [1]. MTA Repair HP is based on the conventional MTA formulation, however it contains calcium tungstate as radiopacifier and the mixing liquid with a plasticizer agent. Among the suggested applications of the said material are: root-end filling, pulp capping, pulpotomy, apexogenesis, apexification as well as the repair of root canal perforations. As the manufacturer's instructions state, this new formula maintains the chemical properties of the original MTA, but additionally shows