10.59987/ads/2014.3.108-114
This study aims to critically summarize the literature about nano-hydroxyapatite. The purpose of this work is to analyze the benefits of using nanohydroxyapatite in dentistry, especially for its preventive, restorative and regenerative applications. We also provide an overview of new dental materials, still experimental, which contain the nanohydroxyapatite 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.
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Nano-hydroxyapatite and its applications in preventive, restorative and regenerative dentistry: a review of literature
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Nano-hydroxyapatite and its applications in preventive, restorative and regenerative dentistry: a review of literature
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                                                                                                    Original article



    Nano-hydroxyapatite and its applications in
    preventive, restorative and regenerative dentistry:
    a review of literature




                                                                                                       i
                                                                                                     al
                                                                                                  on
    Erlind Pepla, MD                                         Key words: nano-HA, preventive, restorative, re-
    Lait Kostantinos Besherat, MD                            generative.
    Gaspare Palaia, DDS
    Gianluca Tenore, DDS
    Guido Migliau, DDS                                       Introduction




                                                                                        zi
                                                             The purpose of this work is to analyze what, up to
    Department of Oral and Maxillo-Facial Sciences,          now, is reported in the literature about the advan-




                                                                          na
    “Sapienza” University of Rome, Italy                     tages of nano-hydroxyapatite in dentistry, especially
                                                             in preventive and restorative dentistry, up to its use in
                                                             oral surgery, such as implantology and periodontal
    Corresponding author:                                    regeneration. We also attempt to provide a broad




                                                               er
    Erlind Pepla                                             overview of the new materials that are being born
    Department of Oral and Maxillo-Facial Sciences           from experimental research, with particular attention
    “Sapienza” University of Rome                            to the materials commonly used in restorative den-
    Viale Regina Elena 278/a                                 tistry, such as composite resins.
    00198 Rome, Italy


                                                        t    Tooth enamel is the most mineralized tissue of human
    E-mail: kledion2004@libero.it
                                                     In      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
    Summary                                                  material is mainly composed by a calcium phosphate
                                                             related to the hexagonal hydroxyapatite, whose chem-
                                        ni
    This study aims to critically summarize the litera-      ical formula is Ca10(PO4)6·2(OH). X-ray energy disper-
    ture about nano-hydroxyapatite. The purpose of           sive spectroscopy (EDS) analysis of enamel and
    this work is to analyze the benefits of using nano-      dentin also indicated the presence in small quantities
                                io

    hydroxyapatite in dentistry, especially for its pre-     of other elements such as Na, Cl and Mg.
    ventive, restorative and regenerative applications.      Hydroxyapatite (HA) is the main component of enam-
    We also provide an overview of new dental materi-        el, which gives an appearance of bright white and
    als, still experimental, which contain the nano-hy-      eliminates the diffuse reflectivity of light by closing
                       iz



    droxyapatite in its nano-crystalline form. Hydroxya-     the small pores of the enamel surface. Hydroxyap-
    patite is one of the most studied biomaterials in the    atite has long been among the most studied bioma-
    medical field for its proven biocompatibility and for    terials in the medical field for both its proven biocom-
          Ed




    being the main constituent of the mineral part of        patibility and for being the main constituent of the
    bone and teeth. In terms of restorative and preven-      mineral part of bone and teeth. Hydroxyapatite is al-
    tive dentistry, nano-hydroxyapatite has significant      so an important source of calcium and phosphate,
    remineralizing effects on initial enamel lesions, cer-   very important for the remineralization of demineral-
    tainly superior to conventional fluoride, and good       ized enamel areas. The inorganic component of all
    results on the sensitivity of the teeth. The nano-HA     the mineralized tissues of the human body is, in fact,
IC




    has also been used as an additive material, in order     made up of a large prevalence of calcium phos-
    to improve already existing and widely used dental       phatesalts. Other inorganic materials such as calci-
    materials, in the restorative field (experimental ad-    um carbonates and sulphates are present in smaller
    dition to conventional glass ionomer cements, that       quantities too; in particular hydroxyapatite represents
C




    has led to significant improvements in their me-         60-70% and 90% in weight of bone and enamel re-
    chanical properties). Because of its unique proper-      spectively. The recently developed interest for nan-
    ties, such as the ability to chemically bond to bone,    otechnology in many fields, is producing interesting
    to not induce toxicity or inflammation and to stimu-     and imminent applications in dentistry for nano-hy-
©




    late bone growth through a direct action on os-          droxyapatite, which presents crystals ranging in size
    teoblasts, nano-HA has been widely used in peri-         between 50 and 1000 nm. The nano-hydroxyapatite
    odontology and in oral and maxillofacial surgery.        has a strong ability to bond with proteins, as well as
    Its use in oral implantology, however, is a widely       with fragments of plaque and bacteria, when con-
    used practice established for years, as this sub-        tained in toothpastes. This ability is due to the size of
    stance has excellent osteoinductive capacity and         nanoparticles, which considerably increase the sur-
    improves bone-to-implant integration.                    face area to which proteins can bind. Besides, nano-

    108                                                                        Annali di Stomatologia 2014; V (3): 108-114
    Nano-hydroxyapatite and its applications in preventive, restorative and regenerative dentistry: a review of literature


    hydroxyapatite also acts as filler because it repairs            many dental structures. In this study the attention
    small holes and depressions on enamel surface, a                 was paid to the search for materials to be added to
    function enhanced by the small size of the particles             common glass ionomer cements available on the
    that compose it. The Japanese company Sangi Co.                  market, Fuji II GC, in order to improve its mechanical
    Ltd was the first to take an interest in hydroxyapatite,         properties.




                                                                                                               i
    after purchasing the rights ifrom NASA (U.S. Nation-             Nano-hydroxy and fluorapatite have been synthe-




                                                                                                             al
    al Aeronautics and Space Authority) in 1970. The as-             sized using a sol-gel technique in an ethanol base.
    tronauts, in fact, lost minerals from the teeth and              The results showed that after 1 and 7 days, the nano-
    bones in the absence of gravity, and NASA proposed               HA/fluorapatite added to cements howed greater




                                                                                                          on
    a synthetic hydroxyapatite as a repairing material.              hardness to compression (CS) 177-179 MPa, a high-
    The Sangi Co. Ltd had the idea in 1978 to launch                 er hardness to diametrical tension (DTS) 19-20 MPa
    toothpaste that could repair the tooth enamel, which             and a higher hardness to biaxial flexibility (BFS) 26-
    contains for the first time nano-hydroxyapatite                  28 MPa, compared to the control group (160 MPa in
    (Apadent). In 2006, the first toothpaste containing              CS, 14 MPain DTS and 18 MPa in BFS) (Tab. 1).




                                                                                                 zi
    synthetic hydroxyapatite biomimetic as an alternative            Therefore, glass ionomers containing nano-bioceram-
    to fluoride for the remineralization and repair of tooth         ics are very promising restorative dental materials
    enamel appeared in Europe. The biomimetic hydrox-                with improved mechanical properties and strong bind-




                                                                                   na
    yapatite function is to protect the teeth with the cre-          ing to dentin, and may very soon replace GIC current-
    ation of a new layer of synthetic enamel around the              ly on the market. From these studies it seems to
    tooth, rather than hardening the existing layer with             emerge, in fact, an unmistakable statistical datum:
    fluorine, that chemically changes into calcium                   modified GICs with the above listed substances pos-




                                                                        er
    halophosphate [Ca5(PO4)3F].                                      sess much higher capacity than traditional materials.
    In its granular form, hydroxyapatite is currently used in        Moshaverina et al. (2008) (2), have extensively stud-
    clinical dental practice to reconstruct periodontal bone         ied the effects of incorporating hydroxyapatite and
    defects, to the fill bone defects after cystectomy, after        fluorapatite (FA) in a conventional glass ionomer ce-
    apicoectomy, after the loss of dental implants and to


                                                             t       ment (Fuji II GC). The addition of synthesized nano-
    increase of the thickness of atrophic alveolar ridges.
    Shaped blocks of hydroxyapatite are especially used
                                                          In         HA and FA in Fuji II improves the mechanical proper-
                                                                     ties (to compressive, diametral tensile and biaxial
    in maxillofacial surgery (bone defects after trauma, os-         flexural forces) of the resulting cement and its bond-
    teotomies and reductive stabilization, reconstruction            ing strength to dentin. These bioceramics are, there-
    of facial skeleton, replacement of parts of orbital and          fore, considered promising additives for glass
                                                  ni
    maxillary bone). Blocks, as well as granular powder,             ionomer cements used as restorative materials. How-
    can also be used in pre-prosthetic surgery to increase           ever, perhaps due to the low solubility value of FA,
    the thickness of the alveolar ridge.                             the FA-containing samples showed very high values,
                                    io

    Studies on biocompatibility have shown that hydroxy-             after 7 and 30 days, in both mechanical properties
    apatite chemically binds to bone and induces no phe-             and binding tests, as compared to compounds con-
    nomena of toxicity nor inflammatory, local or sys-               taining HA and to GIC.
    temic. Some researches show that the hydroxyap-                  In recent years, attention has focused towards the
                           iz



    atite, unlike tricalcium phosphate, doesn’t undergo re-          synthesis of new compounds of nano-HA. This is the
    sorption. Other authors have instead found resorption            case in the study on the remineralizing effects of zinc
    of hydroxyapatite. Thanks to its chemical and crystal-           carbonate nano-HA (ZnCO₃/n-HAP) performed by
          Ed




    lographic affinity with inorganic components that con-           Tschoppe et al. in 2011 (3). In the research, 35
    stitute the bone, hydroxyapatite is able to establish            bovine incisors were taken; from these teeth, 70 ex-
    chemical bonds and to ensure a more rapid integra-               perimental blocks of enamel and 85 samples of
    tion of titanium implants to bone and surrounding tis-           dentin were obtained. A quarter of all the samples
    sues. Numerous studies have highlighted the role of              were coated with a special acid resistant paint, in or-
    hydroxyapatite in facilitating the process of osteointe-         der to act as control group. Enamel lesions were ob-
IC




    gration with or without other polymeric space.                   tained by dipping the blocks in a solution (5l) contain-
                                                                     ing 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 in-
    Analysis of the scientific literature (in restora-               cubator (37° C, BR 6000; Heraeus Kulzer) for 14
C




    tive dentistry)                                                  days. The lesions in dentin were prepared by dipping

    The use of nano-hydroxyapatite as a material that
                                                                     Table 1. The synthesized nano-ceramic particles were in-
    could improve the properties of materials currently
                                                                     corporated in a powder of commercial glass ionomer (Fuji
©




    used in restorative dentistry has been studied.
                                                                     II GIC).
    Moshaverina et al. in 2008 (1) have focused on the
    addition of N-vinylpyrrolidone containing acids, nano-                  Control Group      Nano-HA/Fluoroapatite Group
    hydroxyapatite and fluorapatite to conventional glass
    ionomer cements (GIC). These cements have unique                 CS     160 MPa            177-179 MPa
    properties such as biocompatibility, anticariogenic ac-          DTS    14 MPa             19-20 MPa
                                                                     BFS    18 MPa             26-28 MPa
    tion (due to the release of fluorides) and adhesion to

    Annali di Stomatologia 2014; V (3): 108-114                                                                              109
    E. Pepla et al.


    the samples in a solution containing 0.0476 mMNaF,         years old bulls were analyzed. The teeth, cut into
    2.2 mMCaCl₂ dihydrate, 2.2mM KH₂PO₄, 50 mM                 blocks, were dipped in 8 ml of DS for 72 hours at 37°
    acetic acid and 10 mM KOH, at a pH of 5.0 (37° C)          C. Finally, 70 teeth with a KHN (Knoop Hardness
    for five days. The pH value of the demineralizing so-      Number) value between 171.6 and 204.3 were select-
    lutions was constantly monitored. Afterwards, half of      ed. They were then divided into 10 groups exposed to




                                                                                                         i
    all demineralized surfaces were again covered with         different pH values. Afterward, the blocks were longi-




                                                                                                       al
    paint (baseline control demineralization). The sam-        tudinally sectioned in order to be studied by CSMH.
    ples were divided randomly into five groups (enamel        The data were analyzed using SPSS 13.0 software.
    n=14, dentin n=17) and were placed separately in           The remineralizing effect of nano-HA increased sig-




                                                                                                    on
    remineralizing solution for two and for five weeks. In     nificantly when the pH was lower than 7.0.
    agreement with EN ISO 11609 standards, the respec-         One of the most important variables present in the
    tive toothpastes were diluted in a 1:3 ratio in the rem-   mouth is the variation of pH. The assessment of this
    ineralizing solution, in order to obtain a homogeneous     variable is missing in this study. In another study by




                                                                                          zi
    substance. Commercially available toothpastes con-         Huang et al. of 2009 (5), they analyzed the mineraliz-
    taining ZnCO₃/n-HA or n-HA (all without fluorides)         ing ability of nano-hydroxyapatite in cyclically variable
    were used while the toothpaste containing amino flu-       pH conditions.




                                                                            na
    orides was used for the control group. Then, the sam-      The high availability of calcium and phosphate in
    ples were manually brushed with a soft brush and           these conditions, causes, according to the author, a
    with a minimum pressure; this procedure was per-           positive effect on the remineralization of lesions. This
    formed every day for about 5 seconds and with a            indicates that nano-HA is a better resource of free-
    contact time with the solutions of 115 seconds, a total    Ca, and this is important for the defense from dental




                                                                 er
    time of 120 seconds. We must, however, emphasize           caries and erosion. The largest increase in mineral-
    that this procedure has some significant limitations as    ization was observed in the group with pH 4.0. The
    it is highly operator dependent and difficult to stan-     group with pH 7.0, however, showed the lowest de-
    dardize and empirically assessable. After each brush-      gree of mineralization. An accumulation of many min-
    ing, the samples were rinsed with deionized water for

                                                         t     erals in the lesions and a corresponding reduction of
                                                      In
    10 seconds. Every two days for each group, the solu-       their depth were also observed. The effect of nano-
    tions were changed (250 ml). Finally, sections of 100      HA is better than the effect of micro-HA at pH 7.0 and
    mm were performed and analyzed by means of mi-             at the same concentrations. The concentration of cal-
    croradiography and through an appropriate software         cium in solutions containing nano-HA was greater
    (TMR for Windows 2.0.27.2; Inspektor Research Sys-         than that detected in solutions containing micro-HA.
                                          ni
    tem, Amsterdam, The Netherlands). Thirty samples of        The Ca concentration increase leads to a growth in
    enamel and two of dentin were lost during prepara-         the saturation of oral fluids with HA, favoring the de-
    tion procedures. This complex in vitro study shows         position of apatite minerals in the lesions and eventu-
                                 io


    that toothpastes containing different types of nano-       ally promoting remineralization.
    hydroxyapatite have the same remineralizing capabil-       In terms of dental erosion, it is important to empha-
    ities on enamel and dentin, and those containing fluo-     size that its prevalence is increasing in young chil-
                        iz



    ride have lower capacity than the first. We must, how-     dren and adolescents in developed and in developing
    ever, take into account the limits of an in vitro study,   countries. The main external cause of increased ero-
    as it is far from simulating the conditions present in     sion is a higher consumption of acids in the diet and
          Ed




    the oral cavity.                                           with drinks. In particular the use of sport drinks has
    In the study by Huang et al. of 2009 (4), the authors      recently increased, and these may cause erosion ac-
    analyzed the remineralizing effect of nano-HA on           cording to their acid content. With reference to this, is
    demineralized bovine enamel under cyclical condi-          the interesting Min et al. in 2011 study (6), on nano-
    tions of pH, by the microhardness test, on cross-sec-      hydroxyapatite as an addition to sports drinks. In this
    tions (CSMH) and on surfaces and through polarized         study was examined the possible beneficial effects of
IC




    light microscopy (PLM). Nano-HA and conventional           additioning nano-hydroxyapatite to sports drinks.
    HA (crystals in the order of micrometers, from 0.5 to      Powerade ® (PA) was taken as experimental solution
    2 µm), were obtained by the National Incubation Base       and citric acid was added as acid. They prepared dif-
    of Nano-Biomaterials Industrialization, Sichuan Uni-       ferent solutions with the PA alone and with the addi-
C




    versity. The demineralizing solution (DS) used to cre-     tion of 0.05%, 0.10% and 0.25% nano-hydroxyap-
    ate lesions similar to caries had the following compo-     atite. 20 bovine teeth per group, cut in 3.5 mm x 3.5
    sition: acetic acid50 mM, Ca(NO₃)₂2.2 mM, KH₂PO₄           mm blocks, were treated for 20 minutes three times a
    2.2 mM and NaF 5.3 μM. The pH value of the DS was          day, with 2 h and 40 min of interval between each
©




    adjusted to 4.5 by the addition of a solution of KOH.      treatment. Once the treatment process was finished,
    The remineralizing solution (RS) used under condi-         the samples were thoroughly rinsed with distilled wa-
    tions of cyclical pH contains instead: HEPES 20 mM,        ter. Throughout the rest of the day, when not being
    CaCl₂ 1.5 mM, KH₂PO₄ 0.9 mM, KCl130 mM and                 treated, the teeth were immersed in a solution con-
    NaN₃ 1 mM. The pH value was adjusted to 7.0 with           taining artificial saliva with the following composition:
    KOH. The dissolution of HA products was studied in         gastric mucin 0.22%, KCl 14.93 mM, KH₂PO₄ 5.42
    preliminary experiments. In the study, incisors of 4       mM NaCl 6.51 mM and CaCl₂ dihydrate 1.45 mM.

    110                                                                          Annali di Stomatologia 2014; V (3): 108-114
    Nano-hydroxyapatite and its applications in preventive, restorative and regenerative dentistry: a review of literature


    The cyclic pH process was repeated for 7 days. The               provide rapid relief of dentin hypersensitivity (DH). Us-
    potential erosion was determined by changes in the               ing a double-mask, randomized design, three denti-
    surface microhardness (SMH), and the teeth were an-              frices: 1) containing 8% arginine and 1,450 ppm sodi-
    alyzed with the confocal laser scanning microscopy               um monofluorophosphate; 2) containing 8% strontium
    (CLSM) and with the scanning electron microscope                 acetate and 1,040 ppm sodium fluoride; and 3) con-




                                                                                                               i
    (SEM). The prevention of dental erosions increased               taining 30% microaggregation of zinc-carbonate hy-




                                                                                                             al
    with the concentration of n-HA, and sports drinks con-           droxyapatite nanocrystals were compared after 3-day
    taining 0.25% of the substance have obtained the                 treatment. Participant’s DH was evaluated at baseline
    best results.                                                    and after 3 days using air-blast, tactile, cold water, and




                                                                                                          on
    The consumption of carbonic acid containing drinks is            subjective tests. The final sample consisted of 85 indi-
    the main etiological factor for tooth erosion. An experi-        viduals: 29 received the arginine-based dentifrice
    mental study on 18 permanent teeth dental erosion                (group 1), 27 the strontium acetate-based dentifrice
    caused by beer was conducted by Hangoo et al. (11)               (group 2), and 29 the dentifrice based on zinc-carbon-




                                                                                                 zi
    in 2011 (7). These elements were subsequently treat-             ate hydroxyapatite (group 3). All dentifrices were most-
    ed with a remineralizing substance nano-hydroxyap-               ly effective to reduce DH: the percentage of score re-
    atite based. In the study they primarily measured the            duction from baseline to 3 days was >30% for all tests




                                                                                   na
    microhardness of 18 permanent teeth (Fig. 1). Then               (except for subjective test of group 2). The comparison
    they did second measurement of the teeth, after dip-             among the three dentifrices showed that, after 3 days,
    ping them in a solution containing 40 ml of beer                 there was an improvement in air-blast (mean percent-
    (Behnoush Lemon Delester, Iran) for 5 minutes. The               age of reduction, 39.2% in group 1, 42.0% in group 2,
    time was calculated according to actual studies on the           and 39.2% in group 3), cold water (41.5, 51.8, and




                                                                        er
    permanence of beer in the mouth based on the                     50%), tactile (50.3, 40.1, and 33.8%), and subjective
    amount drunk daily. The average microhardness pri-               (33.1, 17.4, and 31.4%) test scores, with differences
    mary values – i.e. prior to any type of manipulation of          being significant for cold water and subjective tests.
    the teeth –, of the 18 cases was 340.24 ± 25.4²                  For air-blast and tactile tests, there were no significant
    kgf/mm. This value reduced to 314.67 ± 33.89² (sec-

                                                             t       differences across groups at 3 days. Moreover, no sig-
    ond value of microhardness) after immersion in beer;
                                                          In         nificant differences at any test were observed in a sub-
    this is equivalent to 92.5% of the primary value of mi-          set of patients that were followed up to 8 weeks:
    crohardness, and the “t” test analysis shows that this is        all dentifrices were all highly efficacious. This study
    statistically significant (p=6.20). The value of sec-            documents that the three tested dentifrices significantly
    ondary microhardness of the 9 cases in water was                 reduced DH after 3-day treatment, supporting their use
                                                  ni
    312.85 ± 36.79² kgf/mm; that reduces to 310.81 ±                 in clinical practice. To the best of the authors’ knowl-
    31.44² (tertiary value of microhardness) after immer-            edge, this is the first report documenting the rapid re-
    sion in drinkable water. This is equivalent to 99.3%, a          lief from DH of a zinc-carbonate hydroxyapatite denti-
                                    io


    value that is not statistically significant (p=20.6). The        frice. The study by Browing et al. of 2011 (9), finally fo-
    secondary value of microhardness of the 9 cases                  cused attention on the search for a material that could
    dipped in NHAP was 315.18 ± 30.65 kgf/mm. This in-               reduce tooth sensitivity after bleaching. For this pur-
                           iz



    creases to 320.99 ± 24.74² kgf/mm (tertiary value of             pose, nano-hydroxyapatite was tested. It was noted
    microhardness) after immersion in a solution with                that the teeth sensitivity after bleaching increased in
    NHAP. This value is equivalent to 98.2%, which is sta-           the presence of enamel defects. Using a randomized
          Ed




    tistically significant (p=0.012).                                clinical trial, the efficacy of a paste containing n-HA in
    The results of this study demonstrate that there is a            reducing this type of sensitivity was analyzed. A paste
    statistically significant increase in the microhardness          containing n-HA (Renamel AfterBleach, Sangi Co. Ltd,
    of teeth demineralized by beer and then exposed to a             Tokyo, Japan) and a placebo (zero-HA) were randomly
    solution of n-HA.                                                assigned to 42 participants. A 7% hydrogen peroxide
    The aim of the study of Orsini et al. (8) is to evaluate         gel was used for 14 days, in association with a desen-
IC




    the relative abilities of three desensitizing dentifrices to     sitizing paste used immediately for the 5 minutes after-
                                                                     wards. 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 sen-
C




                                                                     sitivity of the teeth were analyzed: percentage of par-
                                                                     ticipants, 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 sen-
©




                                                                     sitivity 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 conclu-
    Figure 1. Microhardness values after demineralization and        sions showed that the group treated with n-HA had
    remineralization.                                                lower sensitivity levels.

    Annali di Stomatologia 2014; V (3): 108-114                                                                              111
    E. Pepla et al.


    Table 2. Number (percentage) of participants with sensitivity to any assessment.

    Number of assessments                        Group                    Participants                      Participants
                                                                          with sensitivity                  without sensitivity
    Baseline                                     Zero-HA                  3 (14%)                           18 (86%)




                                                                                                               i
    First week whitening                         Zero-HA                  14 (67%)                          7 (33%)




                                                                                                             al
    Second week whitening                        Zero-HA                  7 (33%)                           13 (65%)
    First week after whitening                   Zero-HA                  5 (25%)                           15 (75%)
    Baseline                                     n-HA                     2 (10%)                           19 (90%)




                                                                                                          on
    First week whitening                         n-HA                     8 (38%)                           13 (62%)
    Second week whitening                        n-HA                     4 (19%)                           17 (81%)
    First week after whitening                   n-HA                     4 (19%)                           17 (81%)
    n-HA=nano-sizedhydroxyapatite, zero-HA=placebo




                                                                                                zi
    There is no significant difference between the groups during bleaching active phase (chi-square, p=0.06)

    Table 3. Percentage of days associated with sensitivity.




                                                                                   na
    Number ofassessments                       Group                    Days with sensitivity            Days without feeling
    Baseline                                   Zero-HA                  9 (6%)                           138 (94%)
    First week whitening                       Zero-HA                  50 (34%)                         97 (66%)
    Second week whitening                      Zero-HA                  26 (19%)                         114 (81%)




                                                                       er
    First week after whitening                 Zero-HA                  16 (11%)                         124 (89%)
    Baseline                                   n-HA                     7 (5%)                           140 (95%)
    First week whitening                       n-HA                     20 (14%)                         127 (86%)
    Second week whitening                      n-HA                     16 (11%)                         131 (89%)
    First week after whitening                 n-HA


                                                              t         14 (10%)                         133 (90%)
    n-HA=nano-sized hydroxyapatite, zero-HA=placebo
                                                           In
    Participants in the group with n-HA experience significantly more days with sensitivity during the active phase of bleaching
    (chi-square, p=0.001)

    Analysis of the scientific literature (in oral                  microscope (SEM) (XL30; Philips, Eindhoven, The
                                             ni
    surgery)                                                        Netherlands) and by atomic force microscope (AFM)
                                                                    (SPM-9500J3; Shimadzu Corporation, Tokyo,
    Many researchers have focused on the use of nano-               Japan). Male Sprague-Dawley rats, about eight
                                   io


    HA as a co-adjuvant material in oral surgery, espe-             weeks old, were used for this study. The implant
    cially regarding the improvement of the dental implant          sites were prepared at about 10 mm from the distal
    characteristics. In the work of Masahiro et al. of 2012         edge of the femur using a 0.8 mm round bur and
                          iz



    (10), they analyze a new compound with nano-poly-               widening by means of reamers (# ISO 090 and 100).
    morphic crystalline HA applied to microrough titanium           A cylindrical implant that had been machined and
    surfaces through a combination of flame spray and               sandblasted, or sandblasted and coated with HA was
          Ed




    calcination at a low temperature. It was then analyzed          inserted into each hole prepared on the femur. The
    for the biological capacity to increase bone-implant            muscles and skin were then sutured separately with
    integration. The sandblasted microrough titanium im-            absorbable suture. The total number of animals used
    plants and the titanium implants linked to HA and               was78 (54 animals for testing machined implants,
    sandblasted were analyzed via biomechanical histo-              sandblasted, sandblasted and coated with HA; ana-
    morphometric methods in rats.                                   lyzed at 2, 4 and 8 weeks; 24 animals for histological
IC




    In the study the HA used as an addition to implants             analysis for the groups of sandblasted and for sand-
    surface is 55% crystalline and causes an increase of            blasted and coated with HA; analyzed at 2 and 4
    the surface area by 70% when compared to uncoat-                weeks). HA-coated implants showed an increased
    ed microrough surfaces. Furthermore, hydroxyapatite             percentage of bone-implant contact and an increase
C




    is free of impurities, with a calcium/phosphate ratio           in bone volume within 50 μm close to the implant
    of 1.66, thus being equivalent to the stoichiometric            surface. On the contrary, around implants coated
    value of HA. Titanium cylinders (1 mm in diameter               with HA, the bone volume outside the boundary of 50
    and 2 mm long) were obtained from Grade 5 titanium              μm was low. In particular, this study demonstrated
    alloy (Ti-6Al-4V). In order to create the micro-rough-          that nano-crystalline hydroxyapatite is indisputably
©




    ness, titanium samples were sandblasted with alu-               effective in increasing osteoconductivity and inhibit-
    minum oxidemicro-powder. The HA coating was ob-                 ing the infiltration of soft tissue around the implant,
    tained by flame spray on blasted surfaces. The flame            but the effect is quite limited to the microenvironment
    was created by means of acetylene and oxygen, and               around the implant.
    the air was used as a high-speed carrier. All exam-             Ceramic nanoparticles represent an encouraging
    ined surfaces were inspected by scanning electron               class of bone graft substitutes due to their improved

    112                                                                                Annali di Stomatologia 2014; V (3): 108-114
    Nano-hydroxyapatite and its applications in preventive, restorative and regenerative dentistry: a review of literature


    osteointegrative properties. Nano-crystalline hydrox-            (Mv) 18 k Dawas used, derived from BASF (Lud-
    yapatite binds bone and stimulates bone healing en-              wigshafen, Germany). The suspension of nano-hy-
    couraging osteoblastic activity. In the study conduct-           droxyapatite used for the compound was prepared
    ed by Singh et al. in 2012 (11), the authors examine             with a wet method and hydrothermal treatments. The
    the clinical and radiographic results obtained with              proliferation of BMSCs culture on nHA/PA66 mem-




                                                                                                               i
    NCHA bone graft (Sybograf®) associated with colla-               branes has been tested with the MTT method (MTT:




                                                                                                             al
    gen membranes (Periocol ® ), compared with OFD                   [3 - {4,5-dimethylthiazol-2YL}-2,5-diphenyl-2H-tetra-
    (Open Flap Debridement) in the treatment of intra-               zoliumbromide]), and it was found to be higher than
    bony periodontal defects.                                        the negative control group after 1 and 4 days of incu-




                                                                                                          on
    In the work on the comparison between NCHA asso-                 bation and, moreover, did not appear to have any
    ciated bone grafts with collagen membranes and tra-              significant differences after 7 and 11 days of culture.
    ditional OFD technique, Singh et al. (11) designed a             The results of the cell cycle suggest that the mem-
    controlled randomized clinical trial in parallel groups.         brane has no negative influence on cell division. The




                                                                                                 zi
    Eighteen intrabony defects in 14 patients aged be-               membrane has an asymmetric porous structure, in
    tween 25 and 65 years were randomly assigned to a                which pores smaller than 10 microns are distributed
    test group and to a control group. The plaque index,             on one side (microporous layer), while pores ranging




                                                                                   na
    the gingival index, the PPD (probing pocket depth),              between 30 and 200 μM are located on the opposite
    the clinical attachment level (CAL) and the gingival             side (macroporous layer). The microporous layer of
    recession (REC) were recorded at baseline, and were              the membrane prevents the migration of fibrous con-
    reassessed 6 months later (Tab. 4). Furthermore,                 nective tissue in bone defects, being also able to per-
    bone grafts were evaluated through digital software.             meate sufficient nutrients for regenerating tissue. The




                                                                        er
    In the test sites they have placed NCHA bone graft               results show that n-HA/PA66 membrane is a 3D
    with collagen membranes, while in control sites they             porous structure with a dense microporous layer on
    performed only OFD. Recall appointments were set at              one side and with a spongy microporous layer on the
    7 days, 30 days, and then at 3 months and 6 months               other side. In vitro experiments show that nHA/PA66
    (Tab. 4). The association between resorbable mem-

                                                              t      membrane has a good affinity for attaching to BM-
    branes, derived from fish, and NCHA bone grafts is
                                                           In        SCs, and a non-negative effect on cells viability and
    particularly positive as concern to the improvement of           proliferation. The results of in vitro and in vivo studies
    periodontal indices. This work has clearly shown that            indicate that the nHA/PA66 membrane has an excel-
    the additional use of resorbable membranes derived               lent biocompatibility and is indicated for use in guid-
    from fish in combination with NCHA bone grafts is                ed tissue regeneration (GTR) or GBR.
                                                  ni
    clinically, radiographically and statistically significant       Recent studies suggest that nano-crystalline hydrox-
    compared to OFD alone, in terms of reduction of                  yapatite (nano-HA) paste represents a promising
    PPD, CAL gain and percentage of bone filling.                    class of grafting bone substitutes. The study of Kasai
                                    io


    In the study by Qu et al. in 2010 (12), the use of a             et al. of 2008 (13) was conducted to investigate the
    bioactive and osteoconductive composite formed by                proliferation of human periodontal ligament (PDL) in
    nano-hydroxyapatite and polyamide 66 (nHA/PA66)                  cell cultures, in the presence of a nano-HA paste,
                           iz



    was tested, for the creation of a new asymmetric                 and to analyze cells’ associated signal paths. In this
    porous membrane to be used for guided bone regen-                experimental research on the consequences of the
    eration (GBR). Regarding the membranes, they ana-                application of nano-HA in cell culture with human pe-
          Ed




    lyzed the cytotoxicity of the material, the response of          riodontal ligament (PDL), PDL cells were stimulated
    the surfaces to bone formation, the morphology, pro-             with pastes of nano-HA and with an enamel matrix
    liferation and cell cycle progression of bone marrow             derivative (EMD) in a soluble form. The proliferation
    stromal cells (BMSCs) in rat culture. The polygonal              of PDL cells was determined by analyzing the incor-
    and fusiform shape of BMSCs was observed using a                 poration of bromodeoxyuridine (BrdU) in the DNA of
    scanning electron microscope (SEM). In the research              proliferating cells. In order to understand the mecha-
IC




    the PA66 with an average viscosity, molecular weight             nism that underlies the increase in cell proliferation
                                                                     of PDL cells exposed to nano-HA, the phosphoryla-
    Table 4. The plaque index, the gingival index, the PPD           tion of serine/threonine protein kinase Akt was ana-
    (probing pocket depth), the clinical attachment level (CAL)      lyzed using phospho-specific antibodies. The nano-
    and the gingival recession (REC).
C




                                                                     HA paste showed a potential for proliferation, two
                                                                     times lower than EMD, but both substrates signifi-
                           Control group          Test group         cantly increased the proliferation rate (p<0.05) in
    PPD reduction          3.22 ± 1.09 mm         4.33 ± 0.5 mm      comparison to the negative control group. In conclu-
                           p = 0.007              p = 0.007
©




                                                                     sion, it seems that the growth and proliferation rate
    CAL gain               2.77 ± 1.09 mm         3.77 ± 0.66 mm     of PDL cells in the presence of nano-HA paste is me-
                           p = 0.006              p = 0.006          chanically connected to the activation of the receptor
    REC increase           0.55 ± 0.72 mm         0.49 ± 0.52 mm     for the epidermal growth factor receptor (EGFR) and
                           p = 0.025              p = 0.046          its downstream targets ERK1/2 and Akt. In conclu-
    Main gain in filled    2.07 ± 0.67 mm         0.91 ± 0.21 mm
                                                                     sion, these studies suggest that nano-HA paste is a
    radiografic defect     p = 0.007              p = 0.008
                                                                     potent stimulator of cell proliferation, which probably

    Annali di Stomatologia 2014; V (3): 108-114                                                                              113
    E. Pepla et al.


    contributes to the fundamental process of periodon-          this revolutionary substance and a growing number of
    tal tissue regeneration.                                     scientific articles on the subject.


    Final remarks                                                References




                                                                                                                i
                                                                                                              al
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                                                                                                           on
                                                                     cements (GIC). Acta Biomaterialia. 2008;Volume 4, Issue 2,
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                                                                                                 zi
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                                                                                 na
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                                                                    er
    mentary material, in order to improve the dental mate-           centration on remineralization of initial enamel lesion in vitro.
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                                                           t         ro study using bovine enamel. J Dent. 2011 Sep,39(9):629-
                                                        In
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                                                                     35. doi: 10.1016/j.dent.2011.07.001. Epub 2011 Jul 7.
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                                                                     permanent teeth following exposure to soft beer in vitro. Sci-
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                                           ni
    such as nano-HA associated with zinc carbonate,                  P, Bambini F, Putignano A. A 3-day randomized clinical tri-
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                                  io

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                         iz



                                                                 10. Masahiro Y, Takeshi U, Naoki T, Takayuki I, Kaori N, Norio
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                                                                     H, Takeo S, Takahiro O. Bone integration capability of
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          Ed




                                                                     nium implants. Int J Nanomedicine. 2012;7:859-873.
    mation and stimulating bone growth through a direct          11. Singh VP, Nayak DG, Shah D. Clinical and radiographic eval-
    action on osteoblasts, nano-HA has been widely used              uation of Nano-crystalline hydroxyapatite bone graft (Sybo-
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IC




                                                                     compatible evaluation of nano-hydroxyapatite/polyamide 66
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                                                                     composite porous membrane. Int J Nanomedicine. 2010 Aug
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                                                                     9;5:429-35.
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C




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    expect for the coming years an increased interest in             Sep;50(3):279-85.
©




    114                                                                                Annali di Stomatologia 2014; V (3): 108-114
