Nano-Hydroxyapatite Remineralization of In-Situ Induced Enamel Caries

INTRODUCTION
Dental enamel is considered by far the human body’s most rigid tissue, which exists as a means of shielding teeth from mastication and is one of the most important lines of defense against tooth decay. Unfortunately, mature enamel lacks the ability to self-regeneration or heal itself by a cellular repair mechanism when it is subjected to significant mineral loss during dental caries and chemical erosion [1]. However, it is now well recognized that the construction of incipient enamel caries is a reversible process where stages of development alternate with periods of remineralization [2]. The dominating components of the microstructure of human enamel are nano-sized hydroxyapatite particles and micron-structured enamel rods that bundle, by interlacing enamel interrod [3]. The enamel apatite is micro or nanocrystalline of varying composition with several distinct chemical characteristics that include non-stoichiometry, the Ca/P ratio ranges from 1.54 to 1.73, in comparison with 1.67 for synthetic apatite; impurities as structural substituents and surface pollutants, e.g., sodium, magnesium, iron, and other ions [4]. Regardless of the abundance of available materials in the dental markets poised to replace the enamel material, the aim to restore the biological structure of enamel remains a great challenge, enamel is not a living tissue [5]. Conventional commercial materials that are used to substitute enamel include amalgams, composite resins and ceramics and have proved to be substantially different from enamel in terms of chemical composition and crystal construction. Hence the features and appearance of these materials fail to match those of natural enamel. In addition to this, recurrent caries often develops in conjunction with these replacement materials due to unsatisfactory fitting, and weak adhesion to tooth structure. Furthermore, the weakly bonded interface may lead to the shedding of restorative materials, which could spell the failure of restoration and the requirement of further surgery [6-8].
Hydroxyapatite (HA) material is one of the most bioactive and biocompatible resources and is extensively used to coat artificial joints and tooth roots. Nano-HA particles have similarities in morphology, crystal structure and crystallinity to the apatite crystal of tooth enamel [9]. In the last few years, an increasing quantity of studies has revealed that nano-HA has the good potential to remineralize artificial carious lesions and incipient caries lesions [2]. In situ regeneration of hydroxyapatite on demineralized enamel surface could be considered the ideal result of enamel repair, due to the production of an enamel-like crystalline structure layer. For the time being, numerous systems have been tested for the regeneration of enamel material, including electrolytic deposition, hydrothermal synthesis, and the use of surfactants and hydrogen peroxide with varying findings [10-12]. However, these methods are considered extreme and usually undertaken in severe, which cannot be applied during routine clinical procedures [6, 13, 14].
Biomineralization is a process that produces non-living tissues and minerals such as bones, shells and teeth that have specific biological functions and structures [15]. These biominerals are characterized by reproducible and species-specific crystallochemical properties, which include uniform particle sizes, well-defined structures and compositions, high levels of spatial organization, complex morphologies, controlled aggregation and texture and preferential crystallographic orientation, high-order assembly into hierarchical structures [11, 1]. To date, there has been no reported research regarding the nano-HA remineralization of in-situ induced enamel caries, or which manifest the best defense under systems that imitate real-life situations. Therefore, the aim of this study was to evaluate the effect of the size of the HA biomaterial particles used to induce remineralization in human enamels subjected to artificial caries induction

MATERIALS AND METHODS
Collection of teeth samples
Noncarious human first premolars (extracted for orthodontic purposes) were provided by different orthodontic clinics in Mosul and subsequently collected to be used in this study. The use of human molar tissue specimens followed a protocol that was approved by the ethical committee of the college of dentistry at the University of Mosul and agreed upon by the patients involved. The collected specimens had no previous restorations; they were devoid of any developmental anomalies and were taken from patients who had no history of systemic diseases. Samples were cleansed by ultrasonic treatment in deionized water for 20 min after with silicon carbide paper (nos. 600 and 1200) and stored in thymol solution (0.2 wt %) at 4°C before use [16].

Bovine bone preparation
For this study, the raw material of choice was bovine femur bones obtained from a local butcher. As a commencement, the bovine bones were boiled in water for 2 hours as a means of defatting and streamlined removal of macroscopic adhering impurities. Thereafter, the bones were washed and cleaned carefully with water to clear them of all of the attached meat, tendons, bone marrows, and other soft tissues. The bones were then immersed in acetone for two hours and subsequently washed out with water for several times. The process was followed-up by drying said bovine bones to evaporate any absorbed water. After that, the dried bovine bones were crushed into small pieces using a mortar pestle and then milled into smaller particle sizes utilizing a rotary mill (Fritsch, Germany). Finally, the bovine bones were sifted in the range of 45- 125μm using a sieve shaker (Retsch, Germany) [17, 18].

Calcination
In the calcination process, a select amount of 10 g of bone particles was placed in an open alumina crucible and heated up to 1200°C in a furnace (Carbolite, UK) to eliminate organic constituents that remained in the bovine bones, leaving only HAP. [17, 18]. 

Grinding and sieving
The powder that resulted from calcination was crushed and sieved to nano-sized particles by the ball milling machine method.

Characterization of the resultant powder
Fourier Transform Infra-Red Spectroscopy (FTIR)
Bruker ALPHA II FTIR Spectrometer was utilized to analyze the powder samples. FTIR results were undertaken to identify functional groups and their respective vibration modes present in the nano-HA samples. The analysis was carried out in spectra between 400-4000 cm -2 wavelength, to determine the quality and purity of the extracted powder.

X-Ray Diffraction Spectroscopy (XRD) 
The diffractograms were acquired using Bruker D8 Advance Diffractometer, with copper Kα radiation (CuKα 1, 54056 Å), operating at 30 kV and 20 mA. where the spectrum is taken at 2θ: 20°–80° with an incremental step size of 0.04° and an acquisition speed of 1° min-1. The size of the crystals was determined according to the equation Scherrer [19]: 
                                                       
where D is the crystallite size, k=0.9 is a correction factor to account for particle shapes, β is the full width at half maximum (FWHM) of (002) peak, λ is the wavelength of the radiation, and θ is the Bragg angle. 

Preparation of Artificial Caries Lesion.
Teeth samples were decoronated just below the cementoenamel junction using a rotary machine with a diamond disc. The cervical surface of the teeth was covered with an acid-resistant light cure dam. To simulate early caries lesions, tooth enamel was etched with H3PO4 (37 wt %) for 30s.

Preparation of the remineralizing material
For the preparation of remineralizing material, 200 mg of nano HA and organic base compound were poured into 80 ml of ethanol and then ultrasonicated for 5 min. The gel-like material was gathered through centrifugation and washed with ethanol.

Enamel Remineralization
The remineralizing material was dropped onto the enamel surface, the samples were air-dried at 25°C for 5 min, resulting in the formation of a gel-like coated layer on the enamel. Afterwards, the enamel samples were immersed in a modified simulated oral fluid at pH 7.00 ± 0.03 at 37°C and were kept in an incubator for 48 hours. The repaired enamel windows were placed in a water ultrasound bath for 20 min, rinsed with water, and air-dried at 25°C before the examination.

FESEM Analysis
Morphological analysis of the enamel surface changes after the introduction of artificial caries and the application of the remineralizing materials, which was observed in a MIRA3 TESCAN Scanning Electron Microscope, at SEM MAG: 330 Kx, WD: 4.29 mm and View Field: 0.629ⴜm. The electron acceleration voltage was 20 kV. The energy dispersive spectroscopy (EDS) attachment of the electron microscope was also used to determine the atomic composition of various powder samples.

RESULTS AND DISCUSSION
The XRD diffractogram for the prepared non-stoichiometric nano-HA powder samples is presented in Fig. 1. The diffractogram displays multiple diffraction peaks that indicate polycrystalline structures of the nanoparticles that compose the powder. The recognized peak positions in the patterns were recorded and matched with peaks reported in the database of the Joint Committee on Powder Diffraction Standards (JCPDS No. 09–0432). These highs were distinct representatives of pure hexagonal crystalline hydroxyapatite.
The highest peaks belonging to the prepared hydroxyapatite as shown at angles between 30°<2ϴ°<33°, where a high peak is observed; and they are considered a characteristic of the x-ray diffraction pattern, and a “fingerprint” for the hydroxyapatite molecule. Nevertheless, these peaks are composed of two lines corresponding to crystalline hydroxyapatite at 31.517° and 32.642°. In addition, it was found that hydroxyapatite was present in a single base; it means that no other phases corresponding to another calcium phosphate as “α-phase calcium phosphate (α-TCP), β- phase of tricalcium phosphate (β-TCP), tetra calcium phosphate (TTCP)”, among others, were found in the materials obtained. Therefore, it can be concluded that no HA mixing with other crystalline compounds was discerned [20]; moreover, in these diffractograms, a preferential growth in the (211) plane at 2θ = 31.517° is observed. This is a typical characteristic of the HA.
The FTIR analysis was used to study the composition of the prepared HA powder in correspondence with that of standardized HA. Fig. 2 shows the infrared spectra of the prepared HA. In these spectra, bands placed at 629 and 3570 cm-1 corresponding to the hydroxyl (OH-) vibrational groups, can be identified [20,21]. Furthermore, bands at 562, 598, 960 and 1018 cm-1 belonging to phosphate groups (PO4)3 were found. These functional groups are characteristic of hydroxyapatite [22]. Table 1 presents a list of the functional groups, wavelength number and type of vibration mode [23].
Fig. 2 shows the transmittance intensity of the bands at 629 and 3570 cm-1 belonging to OH is lower in the case of the natural HA as compared to synthetic HA. This difference can be attributed to the fact that synthetic HA was obtained by means of reactions between two solutions in an aqueous environment, promoting the formation of a great quantity of this type of function; on the contrary, natural HA was obtained using physical reactions. With this method, the organic material of bones was extracted, to leave only the inorganic part corresponding to different calcium phosphate, mainly the HA. During this process, the material did not remain in contact with any aqueous solution, being the possible cause of no formation of the great number of hydroxyl groups [24]. 
In Fig. 2, bands at 1450 and 1545 cm-1 corresponding to carbonate groups are identified; moreover, in Table 1, the vibrational modes observed in the synthetic HA are listed. These bands are formed, this methodology, a carbonate HA was obtained. This type of HA exhibits a substitution of carbonate ions by hydroxyl ions (A type substitution) and carbonate ions by phosphate ions (B type substitution) [25]. This type of substitution favors the HA powder’s bioactivity because the negative charge carriers initiate and promote the bone type apatite growth in presence of the SBF [26].
The prepared hydroxyapatite SEM images obtained under different magnifications revealed clusters of nano-particles with hexagonal structures and different sizes ranging from 18-34 nm in diameter. These particles are arranged in an aggregation that forms larger molecules due to high surface activities Fig. 3.
In regards, the enamel surfaces that were subjected to artificial caries induction showed a remarkable lost enamel prism core but retained periphery which result in the presence of small depressions due to dissolution of the prism ends. A few focal holes were also discerned. They alternated with smoother surfaces, where the depressions were poorly marked or even absent under a scale bar 20µm as shown in Fig. 4A. At higher magnifications (scale bar 1µm), the depressions appeared wedge-shaped. Detailed examination disclosed globular crystals which appeared less packed, resulting in varying degrees of intercrystalline spaces Fig. 4B.
The repaired enamel had the same morphological texture as native enamel because they were indistinguishable by SEM. The boundary between the repaired and native enamel demonstrates successful epitaxial growth and confirms the formation of a new HA layer. Notably, the resulting HA and the assembled structures in the repaired layer were precisely the same as the native materials. Although both enamel rods and inter rods are HA, they have different orientations in the enamel as shown in Fig. 5.
The EDS results confirm the non-stoichiometric nature of the prepared HD, with a Ca/p ratio of 1.622. The calcium deficiency prepared HA, was attributed to ionic substitutions of different types and amounts of elements like magnesium, strontium, sodium, and silicon. The natural HA found in bones and teeth is non-stoichiometric and displays variable deficiencies in Ca, P and OH. These deficiencies are made up of bionic substitutions of different types and amounts of elements like magnesium, strontium, sodium, and silicon [27].  The presence of these substitutions changes the structure and surface chemistry of HA, which in turn influences the biochemistry of bones, enamel, and dentin. The influence of these ionic species on hard tissues has not been fully elucidated. However, other studies revealed the presence and significance of small concentrations of silicon in osteoid regions of young mice and rats, which indicates the role of silicon in the early stages of bone formation and calcification [28]. Similar in vitro and in vivo studies have also shown the important role of silicon in the growth and development of hard tissues. Comparable studies have revealed the inclusion of magnesium in HA acts as a growth factor and stimulate osteoblast proliferation [29].
Both the enamel rods and inter-rods could be epitaxially grown simultaneously in the repair process as shown in Fig. 5. This coinstantaneous duplication of the HA with differential orientations during the enamel reconstruction means that each individual epitaxial growth process is specific and controllable at the nanoscale, affording a high structural resolution to benefit the construction of materials with complicated architectures. A cross-sectional SEM image reveals that the thickness of the enamel-identical repair layer was approximately 2.0 to 2.8 µm as appeared in Fig. 5, with well-organized and uniform characteristics. All diffraction peaks in the prepared HA and their relative intensities were identical to those of the original enamel HA, implying an identically organized crystallographic structure at the macroscopic level.

CONCLUSION
 Nano-hydroxyapatite had the potential to remineralize initial enamel caries lesions. The precise remineralization of the enamel structure from the nanoscale to the macroscale was achieved. The repair of demineralized tooth enamel could be achieved by this biomimetic tactic since the nano-hydroxyapatite of proper resources could be useful in promoting remineralization.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interests regarding the publication of this manuscript.