Nano-hydroxyapatite and its applications in preventive, restorative and regenerative dentistry: a review of literature
Содержание
This study aims to critically summarize the literature about nano-hydroxyapatite. The purpose of this work is to analyze the benefits of using nano-hydroxyapatite in dentistry, especially for its preventive, restorative and regenerative applications. We also provide an overview of new dental materials, still experimental, which contain the nano-hydroxyapatite in its nano-crystalline form. Hydroxyapatite is one of the most studied biomaterials in the medical field for its proven biocompatibility and for being the main constituent of the mineral part of bone and teeth. In terms of restorative and preventive dentistry, nano-hydroxyapatite has significant remineralizing effects on initial enamel lesions, certainly superior to conventional fluoride, and good results on the sensitivity of the teeth. The nano-HA has also been used as an additive material, in order to improve already existing and widely used dental materials, in the restorative field (experimental addition to conventional glass ionomer cements, that has led to significant improvements in their mechanical properties). Because of its unique properties, such as the ability to chemically bond to bone, to not induce toxicity or inflammation and to stimulate bone growth through a direct action on osteoblasts, nano-HA has been widely used in periodontology and in oral and maxillofacial surgery. Its use in oral implantology, however, is a widely used practice established for years, as this substance has excellent osteoinductive capacity and improves bone-to-implant integration.
Introduction
The purpose of this work is to analyze what, up to now, is reported in the literature about the advantages of nano-hydroxyapatite in dentistry, especially in preventive and restorative dentistry, up to its use in oral surgery, such as implantology and periodontal regeneration. We also attempt to provide a broad overview of the new materials that are being born from experimental research, with particular attention to the materials commonly used in restorative dentistry, such as composite resins.
Tooth enamel is the most mineralized tissue of human body. Its composition is 96 wt.% inorganic material and 4 wt.% organic material and water. In dentin, the inorganic material represents 70 wt.%. This inorganic material is mainly composed by a calcium phosphate related to the hexagonal hydroxyapatite, whose chemical formula is Ca10(PO4)6·2(OH). X-ray energy dispersive spectroscopy (EDS) analysis of enamel and dentin also indicated the presence in small quantities of other elements such as Na, Cl and Mg.
Hydroxyapatite (HA) is the main component of enamel, which gives an appearance of bright white and eliminates the diffuse reflectivity of light by closing the small pores of the enamel surface. Hydroxyapatite has long been among the most studied biomaterials in the medical field for both its proven biocompatibility and for being the main constituent of the mineral part of bone and teeth. Hydroxyapatite is also an important source of calcium and phosphate, very important for the remineralization of demineralized enamel areas. The inorganic component of all the mineralized tissues of the human body is, in fact, made up of a large prevalence of calcium phosphatesalts. Other inorganic materials such as calcium carbonates and sulphates are present in smaller quantities too; in particular hydroxyapatite represents 60–70% and 90% in weight of bone and enamel respectively. The recently developed interest for nanotechnology in many fields, is producing interesting and imminent applications in dentistry for nano-hydroxyapatite, which presents crystals ranging in size between 50 and 1000 nm. The nano-hydroxyapatite has a strong ability to bond with proteins, as well as with fragments of plaque and bacteria, when contained in toothpastes. This ability is due to the size of nanoparticles, which considerably increase the surface area to which proteins can bind. Besides, nano-hydroxyapatite also acts as filler because it repairs small holes and depressions on enamel surface, a function enhanced by the small size of the particles that compose it. The Japanese company Sangi Co. Ltd was the first to take an interest in hydroxyapatite, after purchasing the rights ifrom NASA (U.S. National Aeronautics and Space Authority) in 1970. The astronauts, in fact, lost minerals from the teeth and bones in the absence of gravity, and NASA proposed a synthetic hydroxyapatite as a repairing material. The Sangi Co. Ltd had the idea in 1978 to launch toothpaste that could repair the tooth enamel, which contains for the first time nano-hydroxyapatite (Apadent). In 2006, the first toothpaste containing synthetic hydroxyapatite biomimetic as an alternative to fluoride for the remineralization and repair of tooth enamel appeared in Europe. The biomimetic hydroxyapatite function is to protect the teeth with the creation of a new layer of synthetic enamel around the tooth, rather than hardening the existing layer with fluorine, that chemically changes into calcium halophosphate [Ca5(PO4)3F].
In its granular form, hydroxyapatite is currently used in clinical dental practice to reconstruct periodontal bone defects, to the fill bone defects after cystectomy, after apicoectomy, after the loss of dental implants and to increase of the thickness of atrophic alveolar ridges. Shaped blocks of hydroxyapatite are especially used in maxillofacial surgery (bone defects after trauma, osteotomies and reductive stabilization, reconstruction of facial skeleton, replacement of parts of orbital and maxillary bone). Blocks, as well as granular powder, can also be used in pre-prosthetic surgery to increase the thickness of the alveolar ridge.
Studies on biocompatibility have shown that hydroxyapatite chemically binds to bone and induces no phenomena of toxicity nor inflammatory, local or systemic. Some researches show that the hydroxyapatite, unlike tricalcium phosphate, doesn’t undergo resorption. Other authors have instead found resorption of hydroxyapatite. Thanks to its chemical and crystallographic affinity with inorganic components that constitute the bone, hydroxyapatite is able to establish chemical bonds and to ensure a more rapid integration of titanium implants to bone and surrounding tissues. Numerous studies have highlighted the role of hydroxyapatite in facilitating the process of osteointegration with or without other polymeric space.
Analysis of the scientific literature (in restorative dentistry)
The use of nano-hydroxyapatite as a material that could improve the properties of materials currently used in restorative dentistry has been studied. Moshaverina et al. in 2008 ( 1 ) have focused on the addition of N-vinylpyrrolidone containing acids, nano-hydroxyapatite and fluorapatite to conventional glass ionomer cements (GIC). These cements have unique properties such as biocompatibility, anticariogenic action (due to the release of fluorides) and adhesion to many dental structures. In this study the attention was paid to the search for materials to be added to common glass ionomer cements available on the market, Fuji II GC, in order to improve its mechanical properties.
Nano-hydroxy and fluorapatite have been synthesized using a sol-gel technique in an ethanol base.
The results showed that after 1 and 7 days, the nano-HA/fluorapatite added to cements howed greater hardness to compression (CS) 177–179 MPa, a higher hardness to diametrical tension (DTS) 19–20 MPa and a higher hardness to biaxial flexibility (BFS) 26–28 MPa, compared to the control group (160 MPa in CS, 14 MPain DTS and 18 MPa in BFS) ( Tab. 1 ). Therefore, glass ionomers containing nano-bioceramics are very promising restorative dental materials with improved mechanical properties and strong binding to dentin, and may very soon replace GIC currently on the market. From these studies it seems to emerge, in fact, an unmistakable statistical datum: modified GICs with the above listed substances possess much higher capacity than traditional materials.
Table 1
The synthesized nano-ceramic particles were incorporated in a powder of commercial glass ionomer (Fuji II GIC).
| Control Group | Nano-HA/Fluoroapatite Group | |
|---|---|---|
| CS | 160 MPa | 177–179 MPa |
| DTS | 14 MPa | 19–20 MPa |
| BFS | 18 MPa | 26–28 MPa |
Moshaverina et al. (2008) ( 2 ), have extensively studied the effects of incorporating hydroxyapatite and fluorapatite (FA) in a conventional glass ionomer cement (Fuji II GC). The addition of synthesized nano-HA and FA in Fuji II improves the mechanical properties (to compressive, diametral tensile and biaxial flexural forces) of the resulting cement and its bonding strength to dentin. These bioceramics are, therefore, considered promising additives for glass ionomer cements used as restorative materials. However, perhaps due to the low solubility value of FA, the FA-containing samples showed very high values, after 7 and 30 days, in both mechanical properties and binding tests, as compared to compounds containing HA and to GIC.
In recent years, attention has focused towards the synthesis of new compounds of nano-HA. This is the case in the study on the remineralizing effects of zinc carbonate nano-HA (ZnCO3/n-HAP) performed by Tschoppe et al. in 2011 ( 3 ). In the research, 35 bovine incisors were taken; from these teeth, 70 experimental blocks of enamel and 85 samples of dentin were obtained. A quarter of all the samples were coated with a special acid resistant paint, in order to act as control group. Enamel lesions were obtained by dipping the blocks in a solution (5l) containing 6 pM of MHDP, 3 mMCa Cl2 dihydrate, 3 mM KH2 PO4 and 50 mM acetic acid, at a pH of 4.95, in an incubator (37° C, BR 6000; Heraeus Kulzer) for 14 days. The lesions in dentin were prepared by dipping the samples in a solution containing 0.0476 mMNaF, 2.2 mMCaCl2 dihydrate, 2.2mM KH2PO4, 50 mM acetic acid and 10 mM KOH, at a pH of 5.0 (37° C) for five days. The pH value of the demineralizing solutions was constantly monitored. Afterwards, half of all demineralized surfaces were again covered with paint (baseline control demineralization). The samples were divided randomly into five groups (enamel n=14, dentin n=17) and were placed separately in remineralizing solution for two and for five weeks. In agreement with EN ISO 11609 standards, the respective toothpastes were diluted in a 1:3 ratio in the remineralizing solution, in order to obtain a homogeneous substance. Commercially available toothpastes containing ZnCO3/n-HA or n-HA (all without fluorides) were used while the toothpaste containing amino fluorides was used for the control group. Then, the samples were manually brushed with a soft brush and with a minimum pressure; this procedure was performed every day for about 5 seconds and with a contact time with the solutions of 115 seconds, a total time of 120 seconds. We must, however, emphasize that this procedure has some significant limitations as it is highly operator dependent and difficult to standardize and empirically assessable. After each brushing, the samples were rinsed with deionized water for 10 seconds. Every two days for each group, the solutions were changed (250 ml). Finally, sections of 100 mm were performed and analyzed by means of microradiography and through an appropriate software (TMR for Windows 2.0.27.2; Inspektor Research System, Amsterdam, The Netherlands). Thirty samples of enamel and two of dentin were lost during preparation procedures. This complex in vitro study shows that toothpastes containing different types of nano-hydroxyapatite have the same remineralizing capabilities on enamel and dentin, and those containing fluoride have lower capacity than the first. We must, however, take into account the limits of an in vitro study, as it is far from simulating the conditions present in the oral cavity.
In the study by Huang et al. of 2009 ( 4 ), the authors analyzed the remineralizing effect of nano-HA on demineralized bovine enamel under cyclical conditions of pH, by the microhardness test, on cross-sections (CSMH) and on surfaces and through polarized light microscopy (PLM). Nano-HA and conventional HA (crystals in the order of micrometers, from 0.5 to 2 μm), were obtained by the National Incubation Base of Nano-Biomaterials Industrialization, Sichuan University. The demineralizing solution (DS) used to create lesions similar to caries had the following composition: acetic acid 50 mM, Ca(NO3)22.2 mM, KH2PO4 2.2 mM and NaF 5.3 μM. The pH value of the DS was adjusted to 4.5 by the addition of a solution of KOH. The remineralizing solution (RS) used under conditions of cyclical pH contains instead: HEPES 20 mM, CaCl2 1.5 mM, KH2PO4 0.9 mM, KCl130 mM and NaN3 1 mM. The pH value was adjusted to 7.0 with KOH. The dissolution of HA products was studied in preliminary experiments. In the study, incisors of 4 years old bulls were analyzed. The teeth, cut into blocks, were dipped in 8 ml of DS for 72 hours at 37° C. Finally, 70 teeth with a KHN (Knoop Hardness Number) value between 171.6 and 204.3 were selected. They were then divided into 10 groups exposed to different pH values. Afterward, the blocks were longitudinally sectioned in order to be studied by CSMH. The data were analyzed using SPSS 13.0 software. The remineralizing effect of nano-HA increased significantly when the pH was lower than 7.0.
One of the most important variables present in the mouth is the variation of pH. The assessment of this variable is missing in this study. In another study by Huang et al. of 2009 ( 5 ), they analyzed the mineralizing ability of nano-hydroxyapatite in cyclically variable pH conditions.
The high availability of calcium and phosphate in these conditions, causes, according to the author, a positive effect on the remineralization of lesions. This indicates that nano-HA is a better resource of free-Ca, and this is important for the defense from dental caries and erosion. The largest increase in mineralization was observed in the group with pH 4.0. The group with pH 7.0, however, showed the lowest degree of mineralization. An accumulation of many minerals in the lesions and a corresponding reduction of their depth were also observed. The effect of nano-HA is better than the effect of micro-HA at pH 7.0 and at the same concentrations. The concentration of calcium in solutions containing nano-HA was greater than that detected in solutions containing micro-HA. The Ca concentration increase leads to a growth in the saturation of oral fluids with HA, favoring the deposition of apatite minerals in the lesions and eventually promoting remineralization.
In terms of dental erosion, it is important to emphasize that its prevalence is increasing in young children and adolescents in developed and in developing countries. The main external cause of increased erosion is a higher consumption of acids in the diet and with drinks. In particular the use of sport drinks has recently increased, and these may cause erosion according to their acid content. With reference to this, is the interesting Min et al. in 2011 study ( 6 ), on nano-hydroxyapatite as an addition to sports drinks. In this study was examined the possible beneficial effects of additioning nano-hydroxyapatite to sports drinks. Powerade ® (PA) was taken as experimental solution and citric acid was added as acid. They prepared different solutions with the PA alone and with the addition of 0.05%, 0.10% and 0.25% nano-hydroxyapatite. 20 bovine teeth per group, cut in 3.5 mm × 3.5 mm blocks, were treated for 20 minutes three times a day, with 2 h and 40 min of interval between each treatment. Once the treatment process was finished, the samples were thoroughly rinsed with distilled water. Throughout the rest of the day, when not being treated, the teeth were immersed in a solution containing artificial saliva with the following composition: gastric mucin 0.22%, KCl 14.93 mM, KH2PO4 5.42 mM NaCl 6.51 mM and CaCl2 dihydrate 1.45 mM. The cyclic pH process was repeated for 7 days. The potential erosion was determined by changes in the surface microhardness (SMH), and the teeth were analyzed with the confocal laser scanning microscopy (CLSM) and with the scanning electron microscope (SEM). The prevention of dental erosions increased with the concentration of n-HA, and sports drinks containing 0.25% of the substance have obtained the best results.
The consumption of carbonic acid containing drinks is the main etiological factor for tooth erosion. An experimental study on 18 permanent teeth dental erosion caused by beer was conducted by Hangoo et al. ( 11 ) in 2011 ( 7 ). These elements were subsequently treated with a remineralizing substance nano-hydroxyapatite based. In the study they primarily measured the microhardness of 18 permanent teeth ( Fig. 1 ). Then they did second measurement of the teeth, after dipping them in a solution containing 40 ml of beer (Behnoush Lemon Delester, Iran) for 5 minutes. The time was calculated according to actual studies on the permanence of beer in the mouth based on the amount drunk daily. The average microhardness primary values – i.e. prior to any type of manipulation of the teeth −, of the 18 cases was 340.24 ± 25.4 2 kgf/mm. This value reduced to 314.67 ± 33.89 2 (second value of microhardness) after immersion in beer; this is equivalent to 92.5% of the primary value of microhardness, and the “t” test analysis shows that this is statistically significant (p=6.20). The value of secondary microhardness of the 9 cases in water was 312.85 ± 36.79 2 kgf/mm; that reduces to 310.81 ± 31.44 2 (tertiary value of microhardness) after immersion in drinkable water. This is equivalent to 99.3%, a value that is not statistically significant (p=20.6). The secondary value of microhardness of the 9 cases dipped in NHAP was 315.18 ± 30.65 kgf/mm. This increases to 320.99 ± 24.74 2 kgf/mm (tertiary value of microhardness) after immersion in a solution with NHAP. This value is equivalent to 98.2%, which is statistically significant (p=0.012).
Microhardness values after demineralization and remineralization.
The results of this study demonstrate that there is a statistically significant increase in the microhardness of teeth demineralized by beer and then exposed to a solution of n-HA.
The aim of the study of Orsini et al. ( 8 ) is to evaluate the relative abilities of three desensitizing dentifrices to provide rapid relief of dentin hypersensitivity (DH). Using a double-mask, randomized design, three dentifrices: 1) containing 8% arginine and 1,450 ppm sodium monofluorophosphate; 2) containing 8% strontium acetate and 1,040 ppm sodium fluoride; and 3) containing 30% microaggregation of zinc-carbonate hydroxyapatite nanocrystals were compared after 3-day treatment. Participant’s DH was evaluated at baseline and after 3 days using air-blast, tactile, cold water, and subjective tests. The final sample consisted of 85 individuals: 29 received the arginine-based dentifrice (group 1), 27 the strontium acetate-based dentifrice (group 2), and 29 the dentifrice based on zinc-carbonate hydroxyapatite (group 3). All dentifrices were mostly effective to reduce DH: the percentage of score reduction from baseline to 3 days was >30% for all tests (except for subjective test of group 2). The comparison among the three dentifrices showed that, after 3 days, there was an improvement in air-blast (mean percentage of reduction, 39.2% in group 1, 42.0% in group 2, and 39.2% in group 3), cold water (41.5, 51.8, and 50%), tactile (50.3, 40.1, and 33.8%), and subjective (33.1, 17.4, and 31.4%) test scores, with differences being significant for cold water and subjective tests. For air-blast and tactile tests, there were no significant differences across groups at 3 days. Moreover, no significant differences at any test were observed in a subset of patients that were followed up to 8 weeks: all dentifrices were all highly efficacious. This study documents that the three tested dentifrices significantly reduced DH after 3-day treatment, supporting their use in clinical practice. To the best of the authors’ knowledge, this is the first report documenting the rapid relief from DH of a zinc-carbonate hydroxyapatite dentifrice. The study by Browing et al. of 2011 ( 9 ), finally focused attention on the search for a material that could reduce tooth sensitivity after bleaching. For this purpose, nano-hydroxyapatite was tested. It was noted that the teeth sensitivity after bleaching increased in the presence of enamel defects. Using a randomized clinical trial, the efficacy of a paste containing n-HA in reducing this type of sensitivity was analyzed. A paste containing n-HA (Renamel AfterBleach, Sangi Co. Ltd, Tokyo, Japan) and a placebo (zero-HA) were randomly assigned to 42 participants. A 7% hydrogen peroxide gel was used for 14 days, in association with a desensitizing paste used immediately for the 5 minutes afterwards. A diary was completed daily to note the effect of desensitization and the eventual sensitivity, on a VAS (Visual Analog Scale). Three aspects of the sensitivity of the teeth were analyzed: percentage of participants, number of days and intensity level. Color change was evaluated. For zero-HA and n-HAgroups, respectively, 51 and 29% of participants reported dentinal sensitivity (p=0.06) ( Tab. 2 ). The days of sensitivity were 76 and 36 respectively (p=0.001). The changes in VAS score from baseline have an upward trend in the zero-HA group (p=0.16) ( Tab. 3 ). The color change was equivalent for both groups. The conclusions showed that the group treated with n-HA had lower sensitivity levels.
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