Novel hydroxyapatite nanorods improve anti-caries efficacy of enamel infiltrants

Graphical abstract


  • The presence of more crystalline hydroxyapatite nanorods increases the degree of conversion of enamel infiltrants.

  • Incorporation of hydroxyapatite nanorods in resin infiltrantes induced resistance of surrounding and underlying enamel to recurrent acidic challenges.



Enamel resin infiltrants are biomaterials able to treat enamel caries at early stages. Nevertheless, they cannot prevent further demineralization of mineral-depleted enamel. Therefore, the aim of this work was to synthesize and incorporate specific hydroxyapatite nanoparticles (HAps) into the resin infiltrant to overcome this issue.


HAps were prepared using a hydrothermal method (0 h, 2 h and 5 h). The crystallinity, crystallite size and morphology of the nanoparticles were characterized through XRD, FT-IR and TEM. HAps were then incorporated (10 wt%) into a light-curing co-monomer resin blend (control) to create different resin-based enamel infiltrants (HAp-0 h, HAp-2 h and HAp-5 h), whose degree of conversion (DC) was assessed by FT-IR. Enamel caries lesions were first artificially created in extracted human molars and infiltrated using the tested resin infiltrants. Specimens were submitted to pH-cycling to simulate recurrent caries. Knoop microhardness of resin-infiltrated underlying and surrounding enamel was analyzed before and after pH challenge.


Whilst HAp-0 h resulted amorphous, HAp-2 h and HAp-5 h presented nanorod morphology and higher crystallinity. Resin infiltration doped with HAp-2 h and HAp-5 h caused higher enamel resistance against demineralization compared to control HAp-free and HAp-0 h infiltration. The inclusion of more crystalline HAp nanorods (HAp-2 h and HAp-5 h) increased significantly ( p < 0.05) the DC.


Incorporation of more crystalline HAp nanorods into enamel resin infiltrants may be a feasible method to improve the overall performance in the prevention of recurrent demineralization (e.g. caries lesion) in resin-infiltrated enamel.


Hydroxyapatite [HAp = Ca 10 (PO 4 ) 6 (OH) 2 ] is the most abundant calcium-phosphate mineral present in bone and teeth . HAp can be synthesized as bioceramic and used as solid or porous coating for implants’ surfaces or as filler in bio-composites . HAp possesses interesting properties of biocompatibility, osteoconductivity and bioactivity, which make this material an excellent candidate for therapeutic applications in biomedical science and bioactive therapeutic dentistry . Several methods are currently employed to synthesize HAp crystals , such as reactions in the solid state , co-precipitation , hydrothermal reaction , sol–gel , micro-emulsions and mechanic-synthesis . Each of these syntheses may generate HAp particles with a wide range of particle sizes as well as different chemico-physical characteristics and properties. However, over the last ten years, innovative methods have been used to generate nanoparticles to be incorporated in biomaterials in order to obtain superior physicochemical properties and greater functionalities . Indeed, nanotechnology has the potential to benefit the development of HAp applied in medicine and dentistry . Nano-crystalline HAp can exhibit enhanced bio-reactivity . This superior performance of nano-HAp is due to their similarity to natural HAp found in human hard tissues. Furthermore, nanoscale HAps exhibit higher sintering, densification and tensile strength . However, the bioactivity, biocompatibility and mechanical properties of HAps are determined by their morphology, crystallite size and degree of purity . Therefore, it is highly desirable to synthesize HAps with a controlled-method to achieve precise morphology, crystallinity and size . The hydrothermal synthesis appears to be a feasible alternative to accomplish these targets . Indeed, the outstanding advantage of this route is the capability of inducing the 1D growth, leading to the formation of nanorods, which represents the morphology of HAp in bone and teeth .

Dental caries remains one of the most predominant health disorders in modern society. Caries progression has its threshold generally influenced by the adherence of a specific and complex biofilm onto enamel surface . A cariogenic biofilm utilizes carbohydrates such as sugars as energy source, which are then digested and transformed in catabolic acids (i.e. mainly lactic and acetic acids), which firstly demineralize enamel and subsequently underlying dentin . Likewise most human diseases, dental caries may be easily and more accurately controlled in its initial stages. To date, biomaterials able to arrest caries progression at very early stages of enamel demineralization are resin infiltrants . These materials consist of very low viscosity dimethacrylate-based monomers capable of infiltrating demineralized enamel and paralyzing caries progression. Nevertheless, such materials are unable to prevent further recurrent caries and to remineralize the infiltrated treated enamel .

In dental biomaterials, nano-HAp has shown to be an adequate filler for adhesive resins to improve their adhesion to dental hard tissues and preserve mechanical properties after water aging . However, there is no information so far regarding the use of resin-based enamel infiltrants doped with different types of nano-HAp and on their potential in inhibiting recurrent enamel demineralization.

Thus, the aims of this study were (1) to synthesize and characterize nano-hydroxyapatite (HAp) by using co-precipitation and hydrothermal method in order to regulate crystallinity, crystallite size and morphology of the particles, and (2) to assess their effects on the degree of conversion (DC) and on the protective role of enamel infiltrants containing 10 wt% HAp nanoparticles against recurrent demineralization. The hypotheses tested were: (1) the hydrothermal synthesis creates nanoparticles of hydroxyapatite with resembling shape and size of the enamel HAp; (2) the presence of the HAp nanoparticles in the infiltrant resin would induce difference in the polymerization (DC); (3) the addition of HAp nanoparticles improves the enamel resistance against recurrent demineralization at surrounding, underlying and infiltrated area.

Materials and methods

Synthesis of HAp nanoparticles

A solution of phosphoric acid (0.3 mol L −1 H 3 PO 4 ) was added to a 0.5 mol L −1 CaCl 2 ·H 2 O (99.67% purity, Quimex, Dinamica, São Paulo, Brazil) solution (molar ratio Ca/P = 1.67) under continuous stirring at room temperature. A white precipitate was obtained by the addition of 30% NH 4 OH (99.5% purity, Vetec, São Paulo, Brazil) solution up to reach pH 9 . The white precipitate was washed with distilled water and vacuum filtered. A part of this precipitate represented the specimen HAp0 h. Thereafter, the powder was dispersed in NH 4 Cl 0.1 mol L −1 solution (99.5% purity, Vetec) with pH 9; the weight ratio between the precipitate and the solution was 1:10. The suspensions were placed in a Teflon autoclave covered with stainless steel to receive the hydrothermal treatment at 150 °C for 2 h (HAp2 h) or 5 h (HAp5 h). Finally, the material was vacuum filtered, washed and dried at 80 °C for 4 h and stored in the desiccator . The synthesis followed the equation:

10CaCl 2 + 6H 3 PO 4 + 2NH 4 OH Ca 10 (PO 4 ) 6 (OH) 2 + 18HCl + 2NH 4 Cl

X-ray diffraction

The XRD assay was performed using a X-ray powder diffractometer X’Pert MPD (PANalytical, Westborough, USA) equipped with Co Kα tube ( λ = 1.7890 nm), 40 kV voltage and a 30 mA current in a range of scanning 2 θ = 20–80°. The diffraction patterns were obtained using Bragg–Brentano geometry in continuous mode with speed of 0.5° min −1 and step size of 0.02° (2 θ ). The Rietveld structure refinement was used for interpreting and analyzing the diffraction data using the program Topas Academic . The crystallite size was refined considering an anisotropic macro . For this particular hexagonal case, the Lorentzian and Gaussian components were constrained to be equal for the group planes ( h 0 0) = (0 k 0), and ( h 0 l ) = (0 k l ). All others were refined independently. The background was adjusted by 5 parameters using a Chebyschev polynomial implemented in the software Topas Academic. With this consideration, it was possible to calculate the crystallite size equivalent for the width and length of the HAp nanoparticles. The experiment was performed in triplicate ( n = 3).

FT-IR spectroscopy

The specimens were grounded in an agate mortar and pressed into discs with KBr at a ratio between 1:10 (sample:KBr). After the pressing process, the spectra were recorded in the range 4000–400 cm −1 with 32 scans at 4 cm −1 resolution using the FT-IR equipment FT-IR 8300 (Shimadzu Inc., Tokyo, Japan). This evaluation was performed in triplicate ( n = 3).

Transmission electron microscopy (TEM)

TEM analysis of the HAp nanoparticles was performed using JEOL JEM 1011 (JEOL, Tokyo, Japan) operating at 100 kV and equipped with a CCD camera (Gatan Orius 831). The specimens were diluted in ethanol and deposited onto carbon coated Cu grids. Subsequently, the specimens were dried at 60 °C overnight before analysis. The size distribution curves for HAp2 h and HAp5 h were obtained measuring the width and the length of 200 particles , using the software Image J (US National Institute of Health, Bethesda, USA). On the other hand, for HAp0 h the size was measured only in the largest direction. The whole experiment was undertaken in triplicate ( n = 3).

Experimental infiltrants preparation

A control light-curable resin blend was prepared by mixing 10 wt% urethane dimethacrylate (UDMA), 87 wt% triethylene-glycol-dimethacrylate (TEGDMA), 0.5 wt% camphoroquinone, 1 wt% ethyl 4-dimethylaminebenzoate (coinitiator) and 1.5 wt% diphenyliodonium hexafluorophosphate (polymerization accelerator). In order to prepare the four experimental infiltrants, each nano-HAp previously synthesized were incorporated into the control resin blend (10 wt%) and mixed for 24 h in darkness followed by a final ultrasonic treatment for 10 min to prevent agglomeration and formation of nanoclusters. The four infiltrants created were the neat resin (Control HAp-free), HAp0 h (infiltrants incorporated with nano-hydroxyapatite after no hydrothermal treatment), HAp2 h (infiltrants incorporated with nano-hydroxyapatite after 2 h hydrothermal treatment) and HAp5 h (infiltrants incorporated with nano-hydroxyapatite after 5 h hydrothermal treatment).

Degree of conversion

The degree of conversion (DC) of experimental and control resin infiltrants was surveyed following a previously described protocol . In brief, a small drop (3 μL) of each resin was positioned at a crystal of Attenuated Total Reflection (ATR) in a Fourier-transform infrared spectrophotometer (FT-IR Bruker Spectrometer, Bruker Co., Bremen, Germany). The spectra were assessed before and subsequent to light-curing (40 s; 1100 mW cm −2 , Poly 600, Kavo, Joinville, Brazil). All spectra were obtained in a range of 1800–1500 cm −1 , with 32 scans at 4 cm −1 resolution in transmission mode. The peak height was determined subsequent to baseline subtraction and normalization using the FT-IR software (Opus 7.0, Bruker). DC was calculated using the intensities of aliphatic C C peak at 1635 cm −1 against an internal standard (aromatic carbon–carbon bond peak at 1609 cm −1 ) before and after light-curing, following the formula:

<SPAN role=presentation tabIndex=0 id=MathJax-Element-1-Frame class=MathJax style="POSITION: relative" data-mathml='DC(%)=100−(peakheightofcuredaliphaticC=C)/(peakheightofcuredaromaticC=C)(peakheightofuncuredaliphaticC=C)/(peakheightofuncuredaromaticC=C)×100′>DC(%)=100((peakheightofcuredaliphaticC=C)/(peakheightofcuredaromaticC=C)(peakheightofuncuredaliphaticC=C)/(peakheightofuncuredaromaticC=C)×100)DC(%)=100−(peakheightofcuredaliphaticC=C)/(peakheightofcuredaromaticC=C)(peakheightofuncuredaliphaticC=C)/(peakheightofuncuredaromaticC=C)×100
DC ( % ) = 100 − ( peak height of cured aliphatic C = C ) / ( peak height of cured aromatic C = C ) ( peak height of uncured aliphatic C = C ) / ( peak height of uncured aromatic C = C ) × 100
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Nov 23, 2017 | Posted by in Dental Materials | Comments Off on Novel hydroxyapatite nanorods improve anti-caries efficacy of enamel infiltrants
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