Bossù et al. J Nanobiotechnol (2019) 17:17 https://doi.org/10.1186/s12951-019-0454-6 Journal of Nanobiotechnology RESEARCH Open Access Enamel remineralization and repair results of Biomimetic Hydroxyapatite toothpaste on deciduous teeth: an efective option to fuoride toothpaste Maurizio Bossù1, Matteo Saccucci1*, Alessandro Salucci1, Gianni Di Giorgio1, Erika Bruni2, Daniela Uccelletti2, Maria Sabrina Sarto3, Giuseppe Familiari4, Michela Relucenti4 and Antonella Polimeni1 Abstract Background: Dental caries is a recognized worldwide public health problem. Despite being one of the most efec- tive strategies against dental caries, the excessive use of fuorine may result in a potential risk of developing dental fuorosis especially in children under age of six. The purpose of this work is to analyze a fuorine-free toothpaste con- taining Biomimetic Hydroxyapatite to assess enamel re-mineralizing and repairing properties. Results: The study was performed in vitro and in vivo, comparing the hydroxyapatite toothpaste with two others toothpaste containing diferent fuorine concentrations. The coating efect of the micro-structured Hydroxyapatite nanoparticles reintegrates the enamel with a biomimetic flm reproducing the structure and the morphology of the biologic Hydroxyapatite of the enamel. As demonstrated, the coating is due to the deposit of a new layer of apatite, which presents fewer particles than the natural enamel, not based on the chemical—physical changes occurring in fuorinated toothpastes. Moreover, it shows resistance to brushing as a consequence of chemical bonds between the synthetic and natural crystals of the enamel. Conclusions: The use of Biomimetic Hydroxyapatite toothpastes has proven to be a valuable prevention measure against dental caries in primary dentition since it prevents the risk of fuorosis. Keywords: Biomimetic nanocrystals, Carbonate-hydroxyapatite, Enamel remineralization toothpaste, Zn-carbonate hydroxyapatite Background in permanent teeth is the most widespread condition Dental caries is the most common non-communica- among 313 diseases assessed, afecting 2.3 billion people; ble chronic disease on a global scale, with a signifcant instead, untreated caries in deciduous teeth has shown impact on the public healthcare expenditure as well as a prevalence of 7.8%, afecting 573 million children [1]. the quality of life of each individual. Epidemiological data An oral health study carried out by the WHO in Italy indicates that in most industrialized countries the situa- on children aged 4 and 12, shows a prevalence of caries tion is critical, with 60–90% of the children of school age respectively of 21.6% and 43.1% [2]. Untreated caries in being afected by dental caries. According to the 2015 children is associated with conditions such as distress, Global Burden of Disease (GBD) Study, untreated caries chronic pain, nutrition and sleep disorder, risk of infec- tion and hospitalization. Tese issues impact signifcantly their weight, growth, quality of life. Children’s cognitive *Correspondence: [email protected] development is also hampered due to frequent school 1 Department of Oral and Maxillo-Facial Sciences, Sapienza University absences whose consequences is learning difculties of Rome, Viale Regina Elena 287a, 00161 Rome, Italy Full list of author information is available at the end of the article [3]. Te cost increase for treatments must also be taken © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Bossù et al. J Nanobiotechnol (2019) 17:17 Page 2 of 13 into account [4, 5]. Caries have a multifactorial etiology, as well as the antibioflm activity against Streptococcus characterized by the loss of nonorganic elements of the mutans. Te study was performed in vitro and in vivo, tooth`s hard tissues due to the presence of weak organic comparing the hydroxyapatite toothpaste with two others acids produced by cariogenic bacteria, typically Strep- toothpaste containing diferent fuorine concentrations, tococcus mutans, produced by the metabolism of sim- in order to understand its interaction with the dental tis- ple carbohydrates introduced by our diet. Te increase sues and demonstrating its potential in terms of protect- of acidity in the oral environment triggers the dissolu- ing elements of deciduous teeth. tion of hydroxyapatite crystals of the tooth enamel and spreads calcium and phosphate ions [6]. Saliva becomes Materials and methods oversaturated with calcium and phosphate, contribut- Tree diferent toothpaste were compared: (a) common ing in the decrease of minerals on the enamel surface, toothpaste, (b) commercial toothpaste containing fuo- previously demineralized, and increases the resistance rine 500 ppm; (c) commercial toothpaste containing fuo- to the cariogenic process [7]. In this dynamic process of rine 1400 ppm; (d) toothpaste containing Hydroxyapatite demineralization and re-mineralization, it is essential to nanocrystal (Biorepair®, Coswell S.p.A., Funo, Bologna, reduce the pathologic factors that increase the chance Italy). Biorepair® toothpaste was analyzed by means of of dental caries. Te use of fuorine as the primary pre- HR-SEM microscopy (High Resolution Scanning Elec- vention against dental pathologies is widely documented tron Microscopy). VP-SEM (Variable Pressure Scanning in literature, and its key role is acknowledged since the Electron Microscopy) analysis on dental samples treated water fuorination. Te ability of fuorine to re-mineral- with diferent toothpastes was also carried out. Finally ize dental surfaces is widely demonstrated for both per- antimicrobial properties of Biorepair® were tested. Tis manent and deciduous teeth [8]. For over 50 years it has study was approved by the Ethical Committee at Sapi- been recommended to use products based on fuorine enza University of Rome (no 4681); an informed consent and toothpaste represents by far the most common one forms were signed by the parents of all patients. [9, 10], able to provide higher concentrations of fuo- rine compared to drinking water. Despite being one of Biorepair® preparation protocols for HR‑SEM the most efective strategies which led to a drop in car- Biorepair® toothpaste was previously characterized as ies in industrialized countries [11], it must be taken into reported by Peetsch et al. [17]. For HR-SEM analysis account that water fuorination, fuorine supplements in it was placed on a microscope slide and left in a desic- our diet, the use of toothpastes and topical application of cator for 24 h prior to observation. In order to isolate fuorine may result in a potential risk of developing den- microRepair® particles from the polymeric phase, to tal fuorosis [12]. At an early age, children do not possess study their morphological features, magnetic stirring and full control of the swallowing process and involuntarily centrifugation were used. Tese non-invasive techniques ingest toothpaste during the daily oral hygiene practice, do not afect particles morphology at FE-SEM (Field resulting in a systematic accumulation of fuorine [13, emission scanning electron microscopy) observation. 14]. Although the use of toothpastes with high concentra- Diferent protocols of stirring and centrifugation were tion of fuorine (1500 ppm) allow greater caries control, tested. using these products on children under 6 years of age Biorepair® toothpaste was dissolved in DI water inside increases the chance of fuorosis, which is directly linked a 250 ml becker and then subject to magnetic stirring to the quantity and concentration of fuorine ingested. at 500 rpm for 20 min. Te sample was left overnight Even if statistically, toothpastes with low concentration to sediment. Te supernatant was then retrieved and of fuorine (500 ppm) have not shown a signifcant efect deposited on Si/SiO2 for microscopic analysis (Sam- on caries prevention, they are defnitely safer [6]. Cur- ple Br). Biorepair® toothpaste was dissolved in DI water rent recommendations for children under six indicate inside a 250 ml becker and then subject to magnetic stir- the use of toothpastes with at least 1000 ppm of fuorine ring at 500 rpm for 20 min. Te resulting solution was in pea-sized quantity, under parental supervision [7]. As left overnight to sediment and successively the particles an alternative to fuorine toothpaste, children under the sedimented on the bottom of the becker were recov- age of six may use a toothpaste containing biomimetic ered, eliminating the supernatant, and deposited on Si/ apatite [15, 16]. Such a toothpaste could be highly rec- SiO2 (Sample B1). In order to optimize the material ommended, eliminating the potential risk of fuorosis. to be observed, the toothpaste dissolved in DI water as Te purpose of this work is to analyze a fuorine-free described in (a) and (b) was further subjected to centrif- toothpaste containing Biomimetic Hydroxyapatite (Zinc- ugation at 4000 rpm for 15 min. Te sediment particles Substituted Carbonate-hydroxyapatite, Microrepair), to were then