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	xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">
	<front>
		<journal-meta>
			<journal-id journal-id-type="publisher-id">sv</journal-id>
			<journal-title-group>
				<journal-title>Superficies y vacío</journal-title>
				<abbrev-journal-title abbrev-type="publisher">Superf. vacío</abbrev-journal-title>
			</journal-title-group>
			<issn pub-type="ppub">1665-3521</issn>
			<publisher>
				<publisher-name>Sociedad Mexicana de Ciencia y Tecnología de Superficies y
					Materiales A.C.</publisher-name>
			</publisher>
		</journal-meta>
		<article-meta>
			<article-id pub-id-type="publisher-id">00002</article-id>
			<article-categories>
				<subj-group subj-group-type="heading">
					<subject>Research papers</subject>
				</subj-group>
			</article-categories>
			<title-group>
				<article-title>Effectiveness of a combined silver nanoparticles/fluoride varnish in
					dental remineralization in children: in vivo study</article-title>
			</title-group>
			<contrib-group>
				<contrib contrib-type="author">
					<name>
						<surname>Butrón-Téllez Girón</surname>
						<given-names>C.</given-names>
					</name>
					<xref ref-type="aff" rid="aff1"><sup>1</sup></xref>
					<xref ref-type="corresp" rid="c1">*</xref>
				</contrib>
				<contrib contrib-type="author">
					<name>
						<surname>Mariel-Cárdenas</surname>
						<given-names>J.</given-names>
					</name>
					<xref ref-type="aff" rid="aff1"><sup>1</sup></xref>
				</contrib>
				<contrib contrib-type="author">
					<name>
						<surname>Pierdant-Pérez</surname>
						<given-names>M.</given-names>
					</name>
					<xref ref-type="aff" rid="aff2"><sup>2</sup></xref>
				</contrib>
				<contrib contrib-type="author">
					<name>
						<surname>Hernández-Sierra</surname>
						<given-names>J.F.</given-names>
					</name>
					<xref ref-type="aff" rid="aff2"><sup>2</sup></xref>
				</contrib>
				<contrib contrib-type="author">
					<name>
						<surname>Morales-Sánchez</surname>
						<given-names>J.E.</given-names>
					</name>
					<xref ref-type="aff" rid="aff3"><sup>3</sup></xref>
				</contrib>
				<contrib contrib-type="author">
					<name>
						<surname>Ruiz</surname>
						<given-names>F.</given-names>
					</name>
					<xref ref-type="aff" rid="aff3"><sup>3</sup></xref>
					<xref ref-type="corresp" rid="c2">#</xref>
				</contrib>
			</contrib-group>
			<aff id="aff1">
				<label>1</label>
				<institution content-type="original"> Facultad de Estomatología, Universidad
					Autónoma San Luis Potosí, San Luis Potosí, SLP, México.</institution>
				<institution content-type="orgdiv1">Facultad de Estomatología</institution>
				<institution content-type="orgname">Universidad Autónoma San Luis
					Potosí</institution>
				<addr-line>
					<city>San Luis Potosí</city>
					<state>SLP</state>
				</addr-line>
				<country country="MX">Mexico</country>
			</aff>
			<aff id="aff2">
				<label>2</label>
				<institution content-type="original"> Departamento de Epidemiología Clínica y
					Departamento de Salud Pública, Facultad de Medicina, Universidad Autónoma de San
					Luis Potosí, San Luis Potosí, SLP, México.</institution>
				<institution content-type="orgdiv2">Departamento de Epidemiología
					Clínica</institution>
				<institution content-type="orgdiv1">Facultad de Medicina</institution>
				<institution content-type="orgname">Universidad Autónoma de San Luis
					Potosí</institution>
				<addr-line>
					<city>San Luis Potosí</city>
					<state>SLP</state>
				</addr-line>
				<country country="MX">Mexico</country>
			</aff>
			<aff id="aff3">
				<label>3</label>
				<institution content-type="original"> Facultad de Ciencias, Universidad Autónoma de
					San Luis Potosí, San Luis Potosí, SLP, México</institution>
				<institution content-type="orgdiv1">Facultad de Ciencias</institution>
				<institution content-type="orgname">Universidad Autónoma de San Luis
					Potosí</institution>
				<addr-line>
					<city>San Luis Potosí</city>
					<state>SLP</state>
				</addr-line>
				<country country="MX">Mexico</country>
			</aff>
			<author-notes>
				<corresp id="c1">
					<label>*</label>
					<email>poly97bu@hotmail.com</email>
				</corresp>
				<corresp id="c2">
					<label>#</label>
					<email>facundo@fciencias.uaslp.mx</email>
				</corresp>
			</author-notes>
			<pub-date date-type="pub" publication-format="electronic">
				<day>05</day>
				<month>06</month>
				<year>2020</year>
			</pub-date>
			<pub-date date-type="collection" publication-format="electronic">
				<season>Apr-Jun</season>
				<year>2017</year>
			</pub-date>
			<volume>30</volume>
			<issue>2</issue>
			<fpage>21</fpage>
			<lpage>24</lpage>
			<history>
				<date date-type="received">
					<day>23</day>
					<month>11</month>
					<year>2016</year>
				</date>
				<date date-type="accepted">
					<day>09</day>
					<month>05</month>
					<year>2017</year>
				</date>
			</history>
			<permissions>
				<license license-type="open-access"
					xlink:href="https://creativecommons.org/licenses/by-nc/4.0/" xml:lang="en">
					<license-p>This is an open-access article distributed under the terms of the
						Creative Commons Attribution License</license-p>
				</license>
			</permissions>
			<abstract>
				<title>Abstract:</title>
				<p>Dental caries is an infectious disease that can occur in children, sometimes as
					soon as the teeth eruption starts. Nanotechnology provides effective strategies
					in prevention and treatment of dental caries. The purpose of this study was to
					determine the effectiveness of the addition of silver nanoparticles to a
					fluoride varnish for the remineralization of primary teeth with white spot
					lesions. A trial was carried out in children with dental demineralization (white
					spot lesions) on both maxillary dental teeth. One of the teeth received three
					applications of a fluoride varnish with silver nanoparticles, and the other one,
					three applications of the same varnish without the silver nanoparticles. We
					measured the mineral content of the teeth with a laser cavity detection device
					(DIAGNOdent®) before the first application of the varnishes, and 3 months after
					the last application. The results show significant differences between teeth
					treated with silver nanoparticles. Teeth have been treated with fluoride varnish
					added with silver nanoparticles seemed to enhance dental structure than teeth
					treated with conventional varnish, and this effect was also found between basal
					and final measurements in this group. The fluoride varnish added with silver
					nanoparticles seems to be more effective in the dental remineralization. This
					strategy may be used as a prophylactic measure to avoid development of
					caries.</p>
			</abstract>
			<kwd-group xml:lang="en">
				<title>Keywords:</title>
				<kwd>silver nanoparticles</kwd>
				<kwd>dental biomaterials</kwd>
				<kwd>dental structure</kwd>
			</kwd-group>
			<funding-group>
				<award-group award-type="contract">
					<funding-source>Consejo Nacional de Ciencia y Tecnología</funding-source>
					<award-id>CB-2010-01-153675</award-id>
					<award-id>#274282</award-id>
				</award-group>
			</funding-group>
			<counts>
				<fig-count count="3"/>
				<table-count count="0"/>
				<equation-count count="0"/>
				<ref-count count="18"/>
				<page-count count="4"/>
			</counts>
		</article-meta>
	</front>
	<body>
		<sec sec-type="intro">
			<title>Introduction</title>
			<p>Many strategies have been developed for maintenance of the integrity of the enamel
				structure; the application of fluoride agents in various forms is a proven approach
				that strengthens the enamel structure <sup>[</sup><xref ref-type="bibr" rid="B1"
						><sup>1</sup></xref><sup>]</sup>. Fluorinated varnishes have been used as
				preventive methods for caries development both in the general population, and in
				patients with special needs since they possess the ability to adhere to dental
				surfaces for relatively long time, and they do not require special application
				techniques <sup>[</sup><xref ref-type="bibr" rid="B2"
					><sup>2</sup></xref><sup>-</sup><xref ref-type="bibr" rid="B4"
					><sup>4</sup></xref><sup>]</sup>. These varnishes are also used in the treatment
				of white spot lesions (WSLs), which indicates dental demineralization; it has been
				supported that the early diagnosis or detection of WSLs and the use of non-invasive
				therapies such as fluoride are important strategies for controlling the development
				of dental caries <sup>[</sup><xref ref-type="bibr" rid="B5"
					><sup>5</sup></xref><sup>]</sup>. Silver has been studied in several areas with
				the advent of nanotechnology, they have been synthesized <sup>[</sup><xref
					ref-type="bibr" rid="B6"><sup>6</sup></xref><sup>,</sup><xref ref-type="bibr"
					rid="B7"><sup>7</sup></xref><sup>]</sup>. Silver nanoparticles (AgNPs) have
				previously been used in dental applications, such as in dental restoration,
				endodontic cements for retro-filling, dental implants, and caries-inhibiting
				solutions <sup>[</sup><xref ref-type="bibr" rid="B7"
					><sup>7</sup></xref><sup>-</sup><xref ref-type="bibr" rid="B9"
					><sup>9</sup></xref><sup>]</sup>, since several studies have demonstrated that
				they pursue a bactericidal effect, particularly against Streptococcus mutans,
				without pigmentation of the dental structure and no evidence of cytotoxicity
					<sup>[</sup><xref ref-type="bibr" rid="B10"
					><sup>10</sup></xref><sup>-</sup><xref ref-type="bibr" rid="B13"
					><sup>13</sup></xref><sup>]</sup>. AgNPs incorporation aims to avoid or at least
				to decrease the microbial colonization over dental materials increasing oral health
				levels <sup>[</sup><xref ref-type="bibr" rid="B14"><sup>14</sup></xref><sup>]</sup>.
				The combination of these strategies may confer a better treatment of the WSLs;
				therefore, the purpose of this research was to determine the effectiveness of the
				addition of AgNPs to a fluoride varnish for the remineralization of white spot
				lesions, and to know if the fluoride varnish added with silver nanoparticles can be
				used to improve oral health quality and avoid the development of caries.</p>
		</sec>
		<sec>
			<title>Experimental details</title>
			<sec>
				<title><italic>Synthesis and characterization of silver nanoparticles</italic></title>
			<p>Silver nanoparticles were prepared, as previously reported by Morales <italic>et
					al.</italic>
				<sup>[</sup><xref ref-type="bibr" rid="B15"><sup>15</sup></xref><sup>]</sup>, in an
				aqueous solution by reducing Ag+ ions with albumin solution. The detailed synthetic
				procedure of silver nanoparticles is as follows: An albumin solution was prepared by
				dissolving 0.1 grams of albumin in 30 ml of deionizer water. Another Ag+ ion
				solution was prepared by dissolving 0.05 grams of AgNO3 in 10 ml of deionizer water.
				The particles were prepared by mixing the silver ion solution with the albumin
				solution in a magnetic stirrer and after 5 minutes, 29.9 % ammonium hydroxide was
				added in drop form, producing an adjustment of pH = 11. Albumin and AgNO3 were
				purchased from Sigma-Aldrich; ammonium hydroxide (29.9 %, Fermont) was used without
				any further purification. Mili-Q water (18.2 Ω) was used throughout the experiment.
				In order to corroborate the silver nanoparticles presence, UV-vis absorption spectra
				were obtained with an Oceanoptics S2000-UV-Vis system. Transmission Electron
				Microscopy (TEM) images at 100 kV were carried out using a JEOL-1230.</p>
			</sec>
			<sec>
			<title><italic>Preparation of dental fluoride varnish containing silver
				nanoparticles</italic></title>
			<p>For this preparation, the colloidal solution of AgNPs was filtered and afterward
				washed a minimum of 3 times with ethylic alcohol to remove residual ionic silver and
				other byproducts. After that, nanoparticles were dried to obtain a powder. A mixture
				of silver nanoparticles (powder) and fluoride varnish (Fluor Protector Varnish®,
				Vivadent, Schaan, Liechtenstein) was prepared at 0.1 %wt. In order to assure the
				homogenous incorporation of the AgNPs, the mixture was sonicated for 10 min using an
				ultrasonic sonicator probe (Q500, Qsonica, LLC). Silver concentration was selected
				1000 times more concentrated than the MIC value obtained for these bacteria in
				previous studies <sup>[</sup><xref ref-type="bibr" rid="B16"
					><sup>16</sup></xref><sup>]</sup>.</p>
			</sec>
			<sec>
				<title><italic>Patients and sample collection</italic></title>
			<p>The study was realized in 22 children aged from 1 to 6 years. The criteria of
				inclusion were teeth with demineralization sites; clinically detected as opaque and
				porous white spot lesions on the vestibular surface of anterior and superior primary
				teeth. DIAGNOdent® laser cavity detection pen (KaVo, Biberach, Germany) was used to
				measure the teeth demineralization. Teeth were assigned one tooth of each hemi arc
				to the experimental treatment, which was the fluoride varnish added with 0.1 %
				AgNPs; and the other one to the control treatment, which was the commercial fluoride
				varnish, thus the teeth were our experimental units.</p>
			<p>These varnishes were applied in each teeth vestibular surface with a brush after they
				had been cleaned, dried with gases and isolated with cotton balls. Each tooth was
				subjected to these treatments once a week for a 3 weeks period, and the children
				received standard oral health care, they were followed up for 3 months afterward and
				measured by DIAGNOdent® to evaluate if there was a difference between groups. There
				were any drop-outs from the study.</p>
			<p>Evaluations at all visits were carried out by the same Pediatric Odontologist; who
				was blinded to the treatment assignment. Reproducibility of the measurements
				conducted with the DIAGNOdent® in children by two observers (inter/intra observer
				agreement) was evaluated, performing the Shrout and Fleiss intraclass 3,1
				correlation coefficient. The reproducibility of the measurements; the inter observer
				and intra observer agreement were 100 and 99 %, respectively.</p>
			</sec>
			<sec>
				<title><italic>Statistical analysis</italic></title>
			<p>Statistical analysis was carried out in the statistical package R-3.0.1 (R
				Development Core Team 2013) at a 95 % Confidence Interval (95 % CI). Results are
				expressed as mean ± SD. The assumption of normal distribution of the data was
				confirmed through Shapiro-Wilk tests, and a parametric analysis was performed with a
				paired Student's t-test between basal and final measurements. A value of p &lt; 0.05
				was considered to be statistically significant.</p>
			</sec>
		</sec>
		<sec sec-type="results|discussion">
			<title>Results and discussion</title>
			<p>The absorption spectra of the AgNPs are presented in <xref ref-type="fig" rid="f1"
					>Figure 1</xref>; the spectrum shows the characteristic Surface Plasmon (SP) of
				silver nanoparticles at 416 nm. <xref ref-type="fig" rid="f2">Figure 2</xref> shows
				TEM images, it is possible to observe that AgNPs are monodispersed having
				pseudospherical shapes and an average size of 46 nm.</p>
			<p>
				<fig id="f1">
					<label>Figure 1</label>
					<caption>
						<title>UV-Vis spectra of the silver nanoparticles synthesized.</title>
					</caption>
					<graphic xlink:href="1665-3521-sv-30-02-21-gf1.png"/>
				</fig>
			</p>
			<p>
				<fig id="f2">
					<label>Figure 2</label>
					<caption>
						<title>TEM images of the silver nanoparticles synthesized.</title>
					</caption>
					<graphic xlink:href="1665-3521-sv-30-02-21-gf2.png"/>
				</fig>
			</p>
			<p>DIAGNOdent® basal measurements were similar between the AgNPs varnish treated teeth
				(5.68 ± 1.0 fluorescence intensity) and the commercial varnish treated teeth (5.50 ±
				0.8 fluorescence intensity) (p = 0.53). At the end of the 3-month follow-up, final
				measurements were taken again. Teeth treated with the AgNPs varnish had less mean
				fluorescence intensity than the group treated with commercial varnish (3.68 ± 1.2
				vs. 5.09 ± 1.0, p &lt; 0.001), which signifies an increase of dental
				remineralization in this group. In the experimental treatment group, it was found
				less fluorescence intensity after 3 months of treatment (3.68 ± 1.2), as compared to
				the basal measurement (5.68 ± 1.0, p &lt; 0.001). We did not find significant
				differences in the measurements of the control group (p = 0.051) (<xref
					ref-type="fig" rid="f3">Figure 3</xref>).</p>
			<p>
				<fig id="f3">
					<label>Figure 3</label>
					<caption>
						<title>Changes in dental structure, arbitrary units from basal and final
							measurements are presented for both treatment groups. It was found
							significant differences between final measurements, and between basal
							and final measurements in the experimental group. Asterisks indicate
							p-values under 0.001.</title>
					</caption>
					<graphic xlink:href="1665-3521-sv-30-02-21-gf3.png"/>
				</fig>
			</p>
			<p>New materials are continually being introduced into dental practice. These materials
				require examination to confirm the properties they claim to possess. The application
				of fluoride agents in different forms has been the most effective and frequently
				employed method used in the prevention dental caries <sup>[</sup><xref
					ref-type="bibr" rid="B1"><sup>1</sup></xref><sup>]</sup>.</p>
			<p>These results suggest that the addition of AgNPs to a fluoride varnish seems to
				promote the dental remineralization. These findings indicate that this treatment
				strategy could not only prevent the progression of white spot lesions, but that it
				may revert the process, as evidenced by the reduction in the DIAGNOdent®
				fluorescence intensity after 3 months of treatment.</p>
			<p>Fluoride ions in varnishes have been shown to promote the dental remineralization,
				either alone or in combination with other compounds <sup>[</sup><xref
					ref-type="bibr" rid="B17"><sup>17</sup></xref><sup>]</sup>. Many studies have
				shown that fluoride varnishes can reverse or arrest the demineralization process of
				WSLs when combined with other preventive measures, such as diet control and
				reduction of dental biofilm <sup>[</sup><xref ref-type="bibr" rid="B3"
					><sup>3</sup></xref><sup>]</sup>. Several studies have indicated fluoride
				varnishes to be clinically effective <sup>[</sup><xref ref-type="bibr" rid="B2"
						><sup>2</sup></xref><sup>]</sup> due to their high concentration and safety
				during application <sup>[</sup><xref ref-type="bibr" rid="B17"
					><sup>17</sup></xref><sup>]</sup>. In several studies, the AgNPs have been
				applied to dental materials, which have shown that can change mechanical properties
				of dental materials. AgNPs used in dental materials have good antimicrobial
				properties due to their efficacy in small doses, minimal toxicity, and minimal side
				effects <sup>[</sup><xref ref-type="bibr" rid="B18"
				><sup>18</sup></xref><sup>]</sup>. Therefore we suppose that these particles may act
				synergistically to improve the dental remineralization, although studies to assess
				this claim must be performed, our hypothesis is based that since AgNPs exhibit
				microbial properties; there is a reduction in the number of bacteria adhering to the
				varnish, thus allowing an increase of dental remineralization.</p>
			<p>We believe that our results have children impact; it's primordial to educate them and
				their parents about the risk of developing oral health related issues, and in the
				need of prophylactic measures to avoid this issues. The application of a fluoride
				varnish added with AgNPs seems to be a measure that can be used safely, and it has
				been shown to possess effectiveness for at least three months. The observation of an
				increase in the dental structure treated with fluoride varnish with AgNPs is a
				promising finding for its use as a prophylactic agent for caries prevention.</p>
		</sec>
		<sec sec-type="conclusions">
			<title>Conclusions</title>
			<p>The effectiveness of repeated applications of a fluoride varnish that contains 0.1 %
				46 nm AgNPs in the treatment of white spot lesions in children was demonstrated. We
				found results of dental remineralization after 3 months of application; thus, this
				strategy the addition with AgNPs to the varnish is a promissory treatment for dental
				remineralization and strengthening enamel to protect against dental caries.</p>
		</sec>
	</body>
	<back>
		<ack>
			<title>Acknowledgements</title>
			<p>This study was partially supported by the Consejo Nacional de Ciencia y Tecnología
				(CONACYT) Grant CB-2010-01-153675 and the scholarship #274282.</p>
		</ack>
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