Antibacterial Activity Analysis of Hydroxyapatite Based Materials with Fluorine and Silver Análisis de la Actividad Antibacteriana de Materiales a Base de Hidroxiapatita con Flúor y Plata

This investigation aims to evaluate the antibacterial activity of nanostructured hydroxyapatite based materials doped with silver and fluorine, to be used as a biomaterial with antibacterial activity. Four different formulations were prepared by combustion method: hydroxyapatite, hydroxyapatite-fluorine, hydroxyapatite-silver-fluorine and hydroxyapatite-silver, with 2% of the doping agents. X-ray diffraction technique was used to determine the mineralogy, identifying the presence of Ca5(PO4)3OH, Ca2P2O7, Ag3PO4, AgCa10(PO4)7 Ca5(PO4)3F and CaF2 phases for the studied samples. Scanning electron microscopy was used to study the morphological structure and it showed homogeneous crystallization of the hydroxyapatite and the inclusion of dopant agents. The antibacterial activity was determined using a modified inhibition test zone to observe if the bacteria (E. faecalis) was susceptible to the antimicrobial agent by the appearance of the zone of inhibition on the agar plate. Both the hydroxyapatite-silver and the hydroxyapatite-silver-fluorine materials generated an inhibition zone. It was possible to determine the minimum inhibitory concentration needed to kill most viable organisms after 48 hours of incubation using the broth microdilution method, resulting in 75 μg/ml and 200 μg/ml for the hydroxyapatite-silver and the hydroxyapatite-silver-fluorine formulation, respectively. These materials could be used for the development of new biomaterials that can be used in dental applications.


INTRODUCTION
Hydroxyapatite (HA), Ca 5 (PO 4 ) 3 (OH), is an inorganic calcium phosphate compound present in nature including the human body [1] [2] . Synthetic hydroxyapatite is a biomaterial because it is biocompatible with human tissues and has bioactive properties. Therefore, Nayak [3] says that "it is widely used in various biomedical applications, mainly in orthopedics and dentistry" (p903).
Incorporating dopants into minerals can greatly modify the mineral characteristics related to the crystallization degree or improvement of stability [4] . Adding fluorine to HA produces fluorohydroxyapatite, which is more chemically stable and has a lower solubility than HA alone [5] . HA´s cation exchange rate is very high with heavy metals or ions like Pb 2+ , Ca 2+ , Cu 2+ , Mn 2+ , Co 2+ and Ag + among others [6] . Metallic or ionic silver compounds have been used in a wide variety of products due to their antibacterial activity (including bacteria, viruses and fungi) [4] [7] [8] . According to Kolmas et al. [9] , silver has "strong antibacterial properties of an exceptionally broad spectrum" (p3-4).
At low concentrations, silver cations are microcidal and can be used to treat burns, wounds, ulcers or as coatings of medical devices or implants because it delays the microbial biofilm development [10] . It is important to calculate the silver content in doped hydroxyapatite based materials, according to Kolmas et al. [9] "since it should be high enough to be able to effectively fight microorganism, while at the same time it should also be limited in order that it does not adversely affect the condition of mammalian tissues" (p5). The antimicrobial effect of silver nanostructures material systems occurs in different ways in the microbial cell, such as damage to the cell membrane by destabilization and cell lysis, damage to the subcellular microbial structure, caused by free Ag + ions and generation of reactive oxygen species (ROS) or inactivation of proteins, enzymes and nucleotides, as well as the modification of microbial signal transduction pathways [11] [12] . The antibacterial effects of silver compounds shows less toxicity in human cells by altering metabolic pathways unique to bacteria and inducing apoptosis dependent on particle size [13] . Among all the methods or techniques used to prepare HA, combustion has its advantages because it produces nanocrystalline powders with specific characteristics depending on the combustion heat and gas evolution [4] .

According to Clinical and Laboratory Standards
Institute (CLSI) standards for antimicrobial susceptibility test, there are several ways for determining bactericidal activity of antimicrobial agents such a disk diffusion, broth dilution and agar dilution that measures the inhibitory activity (MIC) of an antimicrobial agent [14] [15] . For routine determinations, the microdilution method is preferred.
Enterocuccus faecalis (E. faecalis) is a microbial indicator of peri-implantitis and endodontic failure, since the bacteria has been found in the tooth root and the surrounding tissue after implant placement [16] [17] . Confocal microscopy studies have demonstrated its persistence in the periapical spaces and the root cementum [18] .
The aim of the present study is to investigate the antibacterial activity of hydroxyapatite and hydroxyapatite based materials doped with silver and fluorine against E. faecalis since it is the most prominent microorganism involved in persistent infections after root canal therapy [18] .

a) Hydroxyapatite based material preparation
In order to prepare the hydroxyapatite based materials with and without silver and fluorine,          A homogeneous crystallization is observed for the HA based material with and without silver and/or fluorine. The brighter zones correspond to silver and fluorine [4] . The grain size of each material happens to be in the nanometric scale.    The HA-Ag-F material has two defined areas, a crystalline one, composed of Ca, P, O and F, and a second interface, between the grain boundaries, composed of Ag, P and O (Figure 7). Figure 8 shows the surface of  Figure 9 shows the inhibition zones formed by the materials after 48 hours of incubation. After seven days, the HA-Ag inhibition zone grew more than the HA-Ag-F, whereas HA and HA-F did not develop an inhibition zone. Figure 10 shows the inhibition zones maintained after seven days.  Figure 11 shows the microdilution trays for the HA-Ag formulation and Figure 12 shows the results for the HA-Ag-F formulation.  HA-Ag formulation shows activity in E. faecalis and a potential use in the treatment for root canals or in other dental applications due to its antibacterial activity.
According to the available literature, these materials could be used to develop new biomaterials that in effect can be use in the health field, particularly in dentistry.