Synthetic Biomimetic Carbonate-Hydroxyapatite

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New biomimetic carbonate-hydroxyapatite nanocrystals (CHA) have been ... In contrast to other mineralized tissues such as bone, dental enamel is not able to ...

Advanced Materials Research Vols. 47-50 (2008) pp 821-824 online at http://www.scientific.net © (2008) Trans Tech Publications, Switzerland

Synthetic Biomimetic Carbonate-Hydroxyapatite Nanocrystals for Enamel Remineralization N. Roveri1,a*, E. Battistella2,b, I. Foltran1,2,c, E. Foresti1,d, M. Iafisco1,2,e, M.Lelli1,f, B. Palazzo1,2,g and L. Rimondini2,h 1Dept.

of Chemistry “G. Ciamician”, University of Bologna, via Selmi 2, 40126 Bologna, Italy of Medical Sciences, University of Eastern Piedmont “Amedeo Avogadro”, via Solaroli 17, 28100 Novara, Italy [email protected], [email protected], c [email protected], [email protected], [email protected], [email protected], [email protected], [email protected] 2Dept.

Keywords: Hydroxyapatite, Biomimetic Nanocrystals, Enamel Remineralization Abstract. New biomimetic carbonate-hydroxyapatite nanocrystals (CHA) have been designed and synthesized in order to obtain a remineralization of the altered enamel surfaces. Synthesized CHA mimic for composition, structure, nano dimension and morphology bone apatite crystals and their chemical-physical properties resemble closely those exhibited by enamel natural apatite. CHA can chemically bound themselves on the surface of natural enamel apatite thanks to their tailored biomimetic characteristics. The remineralization effect induced by CHA represents a real new deposition of carbonate-hydroxyapatite into the eroded enamel surface scratches forming a persistent biomimetic mineral coating, which covers and safeguards the enamel structure. The experimental results point out the possibility to use materials alternative to fluoride compounds which is commonly utilized to contrast the mechanical abrasions and acid attacks. The apatitic synthetic coating is less crystalline than enamel natural apatite, but consists of a new biomimetic apatitic mineral deposition which progressively fills the surface scratches. Therefore the application of biomimetic CHA may be considered an innovative approach to contrast the acid and bacteria attacks. Introduction Dental caries and erosions are common pathologies diffused worldwide. They are due to the action of acids able to solubilize the tooth mineral structures [1-3]. The main cause of this partial dissolution of the enamel surface is the solubility of hydroxyapatite (HA) at low pH [4]. In contrast to other mineralized tissues such as bone, dental enamel is not able to repair themselves when affected by specific dental pathologies such as caries, demineralization, abrasion or fractures because of lack of cells (Fig. 1). Therefore, nowadays, the only way to restore the damaged enamel is to provide a restoration with synthetic materials. Preventive approaches using fluoride compounds are considered the best way to reduce the enamel dissolution. In fact fluoride ions are able to convert the surface of natural apatite into partially fluoride substituted hydroxyapatite. In this way, fluoride compounds reduce apatite solubility [5]. Hydroxyapatite [Ca10(PO4)6(OH)2], representing the most abundant inorganic phase of bone, and dental hard tissues, has been widely studied as bone filler and prosthetic coating just because of its biocompatibility and osteo-conductivity [6]. Poorly crystalline HA nanocrystals have excellent biological properties, lack of toxicity and inflammatory and immunologic responses. In addition they show bioreabsorption properties under physiological conditions. This property can be modulated by ion substitution and crystallinity degree achieved implementing innovative synthesises with nano sized crystals control. The aim of the present study is to highlight the effect of nano-CHA on the remineralization of human enamel. The CHA biomimetic characteristics have been tailored by nano-technology.

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Materials and methods

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Synthesis: plate-acicular shaped CHA, about 100 nm in size, were synthesized according to a modification of methods previously reported [7] and patented [8]. CHA was precipitated from an aqueous suspension of Ca(OH)2 (0.17 M) by slow addition of H3PO4 (0.15 M). The reaction mixture was stirred at 37 °C for 12 hours, then the stirring was suspended allowing the deposition of CHA nanocrystals. The fraction of plate-shaped crystals with granular dimensions Figure1: SEM image of demineralized enamel (Original magnification: 4000X) ranging from 100 to 150 µm was selected for the study. Transmission Electron Microscopy (TEM): investigations were carried out using a Philips CM 100 instrument. The powders samples were ultrasonically dispersed in ultra-pure water and then a few droplets of the slurry deposited on holey-carbon foils supported on conventional copper microgrids. Scanning Electron Microscopy (SEM): observations were carried out by a SEM (Zeiss EVO, 40 XVP) using secondary electrons at 25Kv and various magnifications. X-ray diffraction (XRD): diffraction patterns were collected using an Analytical X’Pert Pro equipped with X’Celerator detector powder diffractometer using Cu Kα radiation generated at 40 kV and 40 mA. Infrared spectroscopic analysis (FTIR): spectra were recorded by a Perkin-Elmer Spectrum One FT-IR equipped with a Perkin-Elmer Auto-image microscope. The spectral resolution was 4 cm-1. Surface area determination: measurements were undertaken using a Carlo Erba Sorpty 1750 instrument by measuring N2 absorption at 77 K and adopting the BET procedure [9]. Sample preparation: Slabs of enamel (3x3mm) were cut with diamond disks from interproximal surfaces of premolars extracted for orthodontic reasons and sonicated for 10 min in 50% ethanol in order to removed any debris. The slabs were etched with 37% orthophosphoric acid for 1 min and then washed with distilled water. The slabs were divided into 3 groups of treatment using respectively fluoride or CHA based toothpaste and only water (control). Each slab was brushed three times a day for a period of 15 days. The intervals between brushing Figure 2: TEM image of synthetic CHA sessions were at least 5 hours. Every brushing session has been performed for 30 sec by an electric toothbrush using constant a pressure and a bean sized aliquot of toothpaste. wetted with tap water, closely resembling the in vivo usual tooth-brushing b procedure. After every treatment, the single enamel slab was washed with tap water using a cleaned tooth-brush in order to 20 40 60 remove residual tooth-paste. Toothbrushes were repeatedly Position [2 theta] washed with tap water after every utilization. Results and discussion

Figure 3: XRD pattern of CHA a) and deproteinated bone b)

Biomimetic carbonate-hydroxyapatite nanocrystals have been synthesized according to a modification of methods previously reported [7] and patented [8]. A nearly stoichiometric Ca/P molar ratio of about 1.7 and containing 5±1 wt% of carbonate ions replacing prevalently phosphate groups has been obtained, resembling biological apatites. TEM image of synthetic CHA nanocrystals shows the plate-acicular morphology and an average dimension of 100 nm (Fig. 2). XRD patterns of synthesized CHA show the characteristic diffraction maxima of an apatite single phase (JCPDS 01-074-0565). In figure 3a X-ray diffraction pattern is compared with that collected for natural CHA from deproteinated bone (Fig. 3b). The broadening of the diffraction maxima in

Advanced Materials Research Vols. 47-50

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CHA X-ray diffraction pattern (Fig. 3a) indicates a relatively low degree of crystallinity, which was quantified 48% according to the method previously described [10]. The crystallinity observed was increased in respect with that determined from the XRD pattern of natural apatite of deproteinated bone (28%). The same observations emerged from the comparison of the FTIR spectra of CHA nanocrystals and natural apatite of deproteinated bone (data not shown). Specific surface area of 100 m2g-1 has been determined for CHA, resulting included in the range of the biological HA nanocrystals (75-170 m2g-1). The XRD patterns collected on the surface of enamel slabs after treatment with CHA or fluoride tooth-pastes and water are reported in Figure 4a, 4b and 4c respectively. The XRD diffraction maxima obtained on the surface of enamel slabs treated with tooth-paste containing fluoride appear slightly more sharpened than those obtained on the enamel slabs treated only with water. This observation reveals an increased crystallinity degree probably due to a partial structural conversion of hydroxyapatite into fluoroapatite. On the contrary, the XRD pattern obtained on the surface of enamel slabs treated with CHA shows the broadened diffraction maxima characteristic of the synthetic CHA, revealing its presence on the enamel surface. The CHA not removed by brushing procedures suggests the formation of chemical bonds between the synthetic nano crystals and natural enamel apatitic crystals. These bonds allow the formation of a persistent CHA coating on the enamel surface whose morphology was detected by SEM analysis. The surfaces of the teeth treated with fluoride (Fig. 5b) were not consistently changed respect to that of demineralization by ortophosphoric acid (Fig. 5c). Actually the both interprismatic and prismatic enamel structures appear still evident. On the contrary after treatment of the enamel slabs with CHA the interprismatic and prismatic enamel structures appear to be completely hidden by a thick homogeneous apatitic layer (Fig. 5a).

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Figure 4 XRD pattern of enamel treated with: CHA a), fluoride b) and control c). (* indicates Al holder diffraction maxima)

Figure 5 SEM images of enamel treated with: CHA a), fluoride b) and control c) (Original magnification: 2000X).

Conclusion In the present study we have chemically, physically and morphologically investigated the enamel surface modifications, in vitro, induced by brushing with toothpastes containing fluoride or CHA. The results highlight that biomimetic nanosized patented CHA [8] produces a persistent coating deposition of carbonate-hydroxyapatite on the enamel surface. This coating is less crystalline than native enamel apatite, and consists of a new apatitic mineral deposition which progressively fills the scratches and pits. On the contrary, the surface remineralisation observed on the specimens treated with fluoride, is mainly based on chemical-physical modifications rather than a formation of a new coating.

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The documented CHA biomimetic coating formation, is the first remineralization process corresponding to a real new mineral apatitic deposition in the demineralized area of enamel surface. Acknowledgements Authors thank the COSWELL President Paolo Gualandi for his encouraging enthusiasm in suggesting this research work. References [1] Eccles J D. Dental erosion of non industrial origin. A clinical survey and classification. J Prosthet Dent 1979; 42: 649-653. [2] Arnadottir I B, Sæmundsson S R, Holbrook W P. Dental erosion in Icelandic teenagers in relation to dietary and lifestyle factors. Acta Odontol Scand 2003; 61: 25-28. [3] Nunn J H, Gordon P H, Morris A J, Pine C M, Walker A. Dental erosion—changing prevalence? A review of British National children's surveys. Int J Paed Dent 2003; 13: 98-105. [4] West N X, Maxwell A, Hughes J A, Parker D M, Newcombe R G, Addy M. A method to measure clinical erosion: the effect of orange juice consumption on erosion of enamel. J Dent 1998; 26: 329–335. [5] Featherstone J D B, Glena R, Shariati M, Shields C P. Dependence of in vitro Demineralization of Apatite and Remineralization of Dental Enamel on Fluoride Concentration. J Dent Res 1990; 69: 620. [6] Roveri N, Palazzo B. Hydroxyapatite Nanocrystals as Bone Tissue Substitute. Nanotechnologies for the Life Sciences; 9: 283-307 ed. By Challa S.S. R. Kumar, WILEY-VCH, 2006. [7] Landi E, Tampieri A, Celotti G, Sprio S. Densification behaviour and mechanisms of synthetic hydroxyapatite. J Eur Ceram Soc 2000; 20: 2377-2387. [8] Guaber S.p.A., Gazzaniga G., Roveri N., Rimondini L., Palazzo B., Iafisco M., Gualandi P. EU Patent 005146, 2006. [9] Greg S J, Sing K S (Eds.), Adsorption, Surface Area and Porosity, Academic Press, 1967. [10] Erkmen Z E. The effect of heat treatment on the morphology of D-Gun sprayed hydroxyapatite coatings. J Biomed Mater Res 1999; 48(6): 861-868.