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VOLUME 5 - NUMBER 2 - 2014
Biocompatibility of a new pulp capping cement



 Poggio C., Ceci M., Beltrami R., Degna A., Colombo M., Chiesa M. 
Original Article, 69-75
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Aim. The aim of the present study was to evaluate the biocompatibility of a new pulp capping material (Biodentine, Septodont) compared with reference pulp capping materials: Dycal (Dentsply), ProRoot MTA (Dentsply) and MTA-Angelus (Angelus) by using murine odontoblast cell line and Alamar blue and MTT cytotoxicity tests. Methods. The citocompatibility of murine odontoblasts cells (MDPC-23) were evaluated at different times using a 24 Transwell culture plate by Alamar blue test and MTT assay. Results. The results were significantly different among the pulp capping materials tested. Biocompatibility was significant different among materials with different composition. Conclusions. Biodentine and MTA-based products show lower cytotoxicity varying from calcium hydroxide-based material which present higher citotoxicity.




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Annali di stomatologia
pulp capping materials
Biocompatibility of a new pulp capping cement
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Ann Stomatol (Roma)
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Biocompatibility of a new pulp capping cement
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                                                                                              Original article



    Biocompatibility of a new pulp capping cement




                                                                                                                li
                                                                    na
    Claudio Poggio, MD, DDS1,                            Introduction
    Matteo Ceci, DMD, PhD1,




                                                                  io
    Riccardo Beltrami, DMD, PhD2,                        Direct pulp capping involves the application of a den-
    Alberto Dagna, DMD, PhD,                             tal material to seal communications between the ex-
    Marco Colombo, DMD, PhD1,                            posed pulp and the oral cavity (mechanical and cari-
    Marco Chiesa, DMD, PhD1




                                                               az
                                                         ous pulp exposures) in an attempt to act as a barrier,
                                                         protect the dental pulp complex and preserve its vital-
                                                         ity (1). Inducing hard tissue formation by pulp cells as
    1 Department of Clinical, Surgical, Diagnostic       an ultimate goal of capping material use is widely ac-
      and Pediatric Sciences, Section of Dentistry,




                                                            rn
                                                         cepted (2).
      University of Pavia, Italy                         Several materials such as calcium hydroxide-based
    2 Department of Brain and Behavioural Sciences,
                                                         materials and more recently mineral trioxide aggre-
      University of Pavia, Italy                         gate (MTA) are commonly used for this purpose (3,4).




                                                         te
                                                         Calcium hydroxide is the most popular agent for di-
                                                         rect and indirect pulp capping and maintaining pulp
    Corresponding author:                                vitality, given its ability to release hydroxyl (OH) and
    Claudio Poggio                                       calcium (Ca) ions upon dissolution (5,6). Both clini-
    Department of Clinical, Surgical, Diagnostic
    and Pediatric Sciences
                                                      In cally and histologically it has been found to produce
                                                         satisfactory results in indirect and direct pulp capping,
    Section of Dentistry, Policlinico “San Matteo”,
                                                         because it is capable of stimulating the formation of
    University of Pavia
                                                         tertiary dentin by the pulp. This is documented by ba-
    Piazzale Golgi 3,
                                                         sic research and clinical studies with reported suc-
                                                ni
    27100 Pavia, Italy
                                                         cess rates in excess of 80% for direct pulp capping
    Phone: +39 0382 516257, +39 3398124925
                                                         (7,8). Currently, calcium hydroxide products are the
    Fax: +39 0382 516224
                                                         best documented and most reliable materials for di-
    E-mail: claudio.poggio@unipv.it
                              o

                                                         rect pulp capping and serve as the “gold standard”
                                                         against which new materials have to be tested (9).
                          izi


                                                         Nevertheless, calcium hydroxide has some draw-
    Summary                                              backs. Poor bonding to dentin, material reabsorption,
                                                         high solubility and mechanical instability are among
    Aim. The aim of the present study was to evaluate    them. In addition, the formation of reparative dentine
           Ed




    the biocompatibility of a new pulp capping materi-   may not be due to the bioinductive capacity of the
    al (Biodentine, Septodont) compared with refer-      material but due to a defense mechanism by the pulp
    ence pulp capping materials: Dycal (Dentsply),       induced by the irritant nature of calcium hydroxide
    ProRoot MTA (Dentsply) and MTA-Angelus (An-          (10, 11); the high pH (12.5) of calcium hydroxide sus-
    gelus) by using murine odontoblast cell line and     pensions causes liquefaction necrosis at the surface
    Alamar blue and MTT cytotoxicity tests.              of the pulp tissue with the formation of a necrotic lay-
    C




    Methods. The citocompatibility of murine odonto-     er at the material-pulp interface (7).
    blasts cells (MDPC-23) were evaluated at different   Dycal (Dentsply) is a self-setting radiopaque calcium
CI




    times using a 24 Transwell culture plate by Ala-     hydroxide-based material used both as a pulp cap-
    mar blue test and MTT assay.                         ping agent and as a liner under restorations, cements
    Results. The results were significantly different    and other base materials. Its toxicity to pulp cells is
    among the pulp capping materials tested. Bio-        well documented (12, 13).
    compatibility was significant different among ma-    Portland cements, commonly named mineral trioxide
    terials with different composition.                  aggregate (MTA) cements (such as ProRoot MTA,
©




    Conclusions. Biodentine and MTA-based prod-          MTA-Angelus, Tech Biosealer and others), are thera-
    ucts show lower cytotoxicity varying from calci-     peutic, endodontic repair calcium silicate materials in-
    um hydroxide-based material which present high-      troduced at first as a grey cement (14). These materi-
    er citotoxicity.                                     als promote the proliferation/differentiation of human
                                                         dental pulp cells (15-17) and show calcified tissue-
    Key words: biocompatibility, MTT test, murine        conductive activity with the ability to encourage new
    odontoblast, pulp capping materials.                 hard tissue formation in terms of dentine bridge devel-

    Annali di Stomatologia 2014; V (2): 69-76                                                                  69
    C. Poggio et al.


    opment over the exposed pulp (18,19). Compared to                   of catalyst paste and base paste. ProRoot MTA and
    calcium hydroxide materials, MTA has an enhanced                    MTA-Angelus, composed of white Portland cement
    interaction with dental pulp tissue (15) with less pulp             and bismuth oxide (31, 32), were prepared following
    inflammation and limited pulp tissue necrosis (18, 20).             the manufacturer’s instructions. Biodentine consists
    Several new calcium silicate-based materials have re-               of a powder in a capsule and liquid in a pipette. The




                                                                                                                                   li
    cently been developed (21-23), aiming to improve                    powder was mixed with the liquid in a capsule in the
    some MTA drawbacks such as its difficult handling                   triturator for 30 seconds. Once mixed, Biodentine




                                                                         na
    property (24) and long setting time (14). Biodentine                sets in about 12 minutes. During the setting of the ce-
    (Septodont) is among these materials and it is claimed              ment calcium hydroxide was formed.
    to be used as a dentine restorative material in addition
    to endodontic indications similar to those of MTA. This




                                                                       io
    agent is characterized by the release of calcium hy-                Odontoblast cell line culture condition
    droxide in solution (25, 26), which when in contact
    with tissue fluids forms hydroxyapatite (27-29).                    The mouse odontoblast cell line (MDPC-23) was
    As pulp capping materials will be in direct contact                 kindly provided from Dr Jacques Eduardo (Dept. Cari-




                                                                    az
    with pulp tissue for long periods of time, their biocom-            ology, Restorative Sciences, Endodontics; University
    patibility is of particular importance. Several methods             of Michigan School of Dentistry). Odontoblast-like cell
    for the determination of biocompatibility of dental ma-             line (MDPC-23) is recommended for application to in
    terials have been recommended, but the analysis of                  vitro studies concerning the biocompatibility of dental




                                                                 rn
    in vitro cellular reactions are generally considered to             materials. Mac Dougall (33), reported that the immor-
    be the initial approach (30). This allows for the basic             talized mouse odontoblast cell line is positioned in
    biological characterization of a material and for analy-            the periphery of the pulp and are the first cells affect-




                                                              te
    sis of the underlying cellular mechanisms.                          ed by dental materials.
    The aim of the present study is to evaluate the bio-                MDPC-23 cells were cultured in DMEM medium
    compatibility of a new pulp capping material (Bioden-               (Biowhittaker, Italy) supplemented with 10% fetal
    tine, Septodont) compared with reference pulp cap-                  bovine serum (FBS), 2% glutamine, 2% sodium pyru-
    ping materials: Dycal (Dentsply), ProRoot MTA
    (Dentsply) and MTA-Angelus (Angelus) by using
                                                           In           vate, 1% amphotericin and 2% (w/v) streptomycin/
                                                                        penicillin at 37 °C in 5% CO2 atmosphere (34). The
    murine odontoblast cell line and Alamar blue and                    cells were routinely detached using a trypsin-EDTA
    MTT test.                                                           solution for 2 minutes at 37°C, and resuspended in
                                                                        DMEM medium.
                                             ni
                                                                        For the cytotoxicity tests, MDPC cells were deposited
    Materials and methods                                               in the lower chamber of the 24 well culture plate and
                                                                        left for 4 hours at 37°C before any experiment.
    The following materials were used: Dycal, ProRoot
                              o

    MTA, MTA-Angelus and Biodentine. The components
    of each material and its manufacturer are reported in               Cytotoxicity tests
                          izi


    Table 1.
    Dycal, a two-paste system made of a base paste and                  Cytotoxicity tests were performed with the Transwell
    a catalyst paste (13), was prepared following the                   insert (Sigma-Aldrich, St. Louis, MO) methodology
    manufacturer’s instructions by mixing equal amounts                 and the immortalized mouse odontoblast cell line MD-
            Ed




    Table 1. Characteristics of tested materials.

    Material           Components                                                            LOT            Manufacturer
    Dycal              Base paste: (1,3-butylene glycol disalicylate, zinc                   120717         Dentsply Tulsa Dental,
    C




                       oxide, calcium phosphate, calcium tungstate, iron                                    Johnson City, TN, USA
                       oxide pigments).
CI




                       Catalyst paste: (calcium hydroxide, N-ethyl-o/p-toluene
                       sulphonamide, zinc oxide, titanium oxide, zinc stearate,
                       iron oxide pigments).
    ProRoot MTA        Powder: calcium phosphate, calcium oxide, silica, bismuth oxide.      12001879       Dentsply Tulsa Dental,
                       Liquid: distilled water                                                              Johnson City, TN, USA
©




    MTA-Angelus        Powder: potassium oxide, aluminum oxide, sodium oxide, iron           24120          Angelus, Londrina,
                       oxide, sulfur trioxide, calcium oxide, bismuth oxide, magnesium                      PR, Brazil
                       oxide, potassium sulfate, sodium sulfate, silica.
                       Liquid: distilled water
    Biodentine         Powder: tricalcium silicate, dicalcium silicate, calcium carbonate,   B06562         Septodont, Saint-
                       calcium oxide, iron oxide, zirconium oxide.                                          Maur-des-Fosses,
                       Liquid: calcium chloride, hydro soluble polymer.                                     France


    70                                                                                       Annali di Stomatologia 2014; V (2): 69-76
    Biocompatibility of pulp capping material


    PC-23. The advantage of using a non direct contact           ostasis. The loss of plasma membrane asymmetry is
    test for the evaluation of the dental material citotoxici-   an early event in apoptosis, independent of cell type,
    ty is related to the fact that cells and materials are       resulting in the exposure of phosphatidylserine (PS)
    usually separated (35).                                      residues at the outer plasma membrane leaflet (36).
    Cytotoxicity of the four pulp-capping materials was as-      PSVue reagents are a family of fluorescent probes




                                                                                                                      li
    sessed with MDPC-23 cells grown in the lower cham-           containing a bis(zinc 2+ dipicolylamine) group (Zn-
    ber of a 24-mm diameter Transwell plate with a 0,3           DPA), a motif that has been found to bind with high




                                                                     na
    mm pore size polycarbonate membrane (Sigma) (35).            affinity to surfaces enriched with anionic phospho-
    In order to standardize the samples, for the various         lipids, especially phosphatidylserine (PS) exposed on
    analyzes and evaluations, the materials were placed          cell membranes. The plate was kept under gentle agi-
    on sterile paper disks of 0.5 cm in diameter. All test       tation for 2 hours at room temperature. After 2 hours,




                                                                   io
    materials were prepared and mixed under sterile hood         the solution of PSVue has been eliminated and the
    following preparation methods recommended by the             washing of the plate has been carried out with abun-
    manufacturer. The excess material was removed us-            dant Buffer-TES. The next step involved the addition
    ing sterile spatula. Dycal, ProRoot MTA, MTA-Angelus




                                                                az
                                                                 of the dye Hoechst 33342, affine to DNA for viable
    and Biodentine were mixed on special glass plates            cells. After 15 minutes the images were acquired us-
    and later will be placed with sterile carrier on paper       ing confocal laser scanning microscope (CLSM) (37).
    disks. The Transwell membrane of the inner chamber,
    filled with the paper disks, was then placed into the




                                                             rn
    lower chamber of the 24 well culture plate each con-         Results
    taining at the bottom 5x104 cells and incubated at 37
    °C in 5% CO2 atmosphere for 24 h, 48 h and 72 h, re-         Cytotoxicity tests




                                                          te
    spectively. In order to improve the search, the per-
    centage of vitality of the cells was evaluated in three      Figure 1 shows the results obtained with the Alamar
    time intervals: 24, 48 and 72 hours. Some wells were         blue test at 24, 48 and 72 hours.
    incubated with only tissue culture media (negative
                                                                 The results obtained to 24 hours show that the higher
    control) whereas others with a 10% dilution of 30%
                                                       In
    H2O2 (positive control). The vitality was assessed by
                                                                 percentage of cell vitality is found in Biodentine
                                                                 (106%), which shows an average of even greater
    Alamar blue test. For a further control, the percentage
                                                                 compared to the negative control cells, while Dycal
    of vitality of murine odontoblasts, at 72 hours, was al-
                                                                 (8.6%) is the material that presents the lowest values,
    so assessed with the MTT assay (bromide 3-(4,5-di-
                                                                 so as to become the minimum value of the range of
                                                ni
    methylthiazol-2-yl)-2, 5-diphenyltetrazolium). The vi-
                                                                 vitality of the pulp-capping materials tested in re-
    tality test to Alamar blue reagent acts as an indicator
                                                                 search. ProRoot MTA and MTA-Angelus both show
    of cell health, determining the reducing power in order
                                                                 good percentage of vitality, which amounted to 95%
    to measure quantitatively the proliferative capacity;
                              o

                                                                 and 93.6% respectively.
    the reagent was added in a ratio of 1:10 to the cell
                                                                 In the assessment performed at 48 hours, dissimilar
    culture and then the cells were kept in the incubator
                          izi


    for 3-4 hours at 37º C. The degree of fluorescence           results emerge between the various materials. Some
    and the relative values of absorbance were then ac-          of them show an improvement of the percentage of
    quired by reading in a spectrophotometer at a wave-          vitality; MTA-Angelus equals the number of cells of
    length of 595 nm. The MTT test is a standard colori-         the negative control and Biodentine presents a cell vi-
           Ed




    metric assay for measuring the activity of enzymes           tality greater than 13% compared to control. Contrari-
    that reduce the MTT to formazan (a salt blue) in the         wise Dycal and ProRoot MTA show a deterioration of
    mitochondria, giving the substance a blue/purple col-        the data.
    or. This reaction is assessed and measured by the            In the assessment at 72 hours, the analysis of the
    spectrophotometric reading of the sample, at a wave-         samples show a worst general behavior of the materi-
    length of 570 nm. Five replicates for each pulp cup-         als which leads to a decrease in the percentage of vi-
    C




    ping material were used for each experiment per-             tality and in the average number of cells. The only ex-
    formed in duplicate.                                         ceptions are Biodentine and Dycal. Biodentine is the
CI




                                                                 material with the best percentage compared to the
                                                                 negative control, thus demonstrating a marked bio-
    Confocal Laser Scanning Microscope (CLSM)                    compatibility. The average number of cells remains
                                                                 stable compared to the previous assessment made at
    Once performed the cytotoxicity test of the different        48 hours with a percentage which stabilizes at 114%.
    materials, the Transwell inserts was removed and the         Dycal demonstrates a slight increase in the number
©




    land was eliminated from the culture plate. After            of cells (6%), with a substantially cytotoxic behavior.
    washing the slides with the buffer Buffer-TES, 250 ml        ProRoot MTA and MTA-Angelus prove to have a
    of 10 mM solution of the fluorescent dye PSVue               slight negative trend, but with good percentage of vi-
    TM480 were added per well, in order to detect the            tality that are stabilized on 71% for both materials.
    presence of apoptotic cells present in the culture.          Figure 2 shows the results of the vitality tests per-
    Apoptosis is defined as programmed, physiological            formed with the MTT assay at 72 hours. The MTT test
    cell death and plays an important role in tissue home-       confirmed the percentage ratios between the various

    Annali di Stomatologia 2014; V (2): 69-76                                                                        71
    C. Poggio et al.


                                                                    after incubation with different pulp capping materials
                                                                    confirmed the cytotoxicity tests results: a few cells
                                                                    were observed in the presence of Dycal, indicating an
                                                                    high level of citotoxicity (Fig. 5) whereas ProRoot
                                                                    MTA (Fig. 6), MTA-Angelus (Fig. 7) and Biodentine




                                                                                                                              li
                                                                    (Fig. 8) did not seem to be cytotoxic.




                                                                        na
                                                                    Statistical analysis

                                                                    As reported in Table 2, after 24 hours the amount of




                                                                      io
                                                                    cells present in contact with MTA-Angelus is not sta-




                                                                   az
    Figure 1. Alamar blue test results at 24, 48 and 72 hours.




                                                                rn
                                                             te
                                                          In
    Figure 2. Results of the vitality test performed with the MTT
                                            ni
    assay at 72 hours.


    materials and between the materials and the posi-
                             o

    tive/negative controls determined with the Alamar               Figure 3. CLSM images of apoptosis assay in the transwell
    blue test.. MTA Angelus shows the best percentage               wells prepared with a 30% solution of hydrogen peroxide
                         izi


    of vitality at all. In general even if the relationships        (positive control).
    between the various materials are similar, there was
    a slight increase in the mean number of cells.
           Ed




    Confocal Laser Scanning Microscope (CLSM)
    images

    After staining with PSVueTM480 and Hoechst 33342
    dyes, the morphological structure of the cells in cul-
    C




    ture was observed with CLSM. The use of fluorescent
    dye PSVueTM480 shows the presence of apoptotic
CI




    cells. The Hoechst 33342 dye acts by binding to the
    DNA of viable cells and coloring the nucleus in blue.
    Figure 3 shows the negative control observations
    (preparation containing only the culture medium)
    while Figure 4 shows the positive control observa-
    tions (prepared with hydrogen peroxide added to the
©




    culture medium). As clearly shown, H2O2 is very cyto-
    toxic and the cells stained with PSVue480™ reagent
    are completely fluorescent in green (Fig. 3). In ab-
    sence of any type of materials, the cells were not
    green fluorescent (Fig. 4) but we could see the nuclei
    stained with Hoechst. Figures 5 - 8 show the images             Figure 4. CLSM images of apoptosis assay in presence of
    acquired for each material. These images obtained               culture medium only (negative control).

    72                                                                                  Annali di Stomatologia 2014; V (2): 69-76
    Biocompatibility of pulp capping material




                                                                                                                                       li
                                                                           na
                                                                         io
                                                                      az
                                                                   rn
    Figure 5. CLSM images of apoptosis assay in the transwell             Figure 7. CLSM images of apoptosis assay in the transwell
    wells prepared with Dycal.                                            wells prepared with MTA-Angelus.




                                                                te
                                                             In
                               o                ni
                           izi
            Ed




    Figure 6. CLSM images of apoptosis assay in the transwell             Figure 8. CLSM images of apoptosis assay in the transwell
    C




    wells prepared with ProRoot MTA.                                      wells prepared with Biodentine.
CI




    Table 2. Mean ± standard deviation of Bonferroni post-hoc test of the different values of cell viability. Different superscript
    letters indicate a statistically significant difference (P <0.001). The same superscript letter indicates a not statistically signifi-
    cant difference (P> 0.001).
©




    Materials                            24 h                                   48 h                                  72 h
    Negative control                     500000 ± 0a                            522000 ± 0d                           466000 ± 0g
    Positive control (H2O2)              37000 ± 2738b                          25000 ± 1850e                         18000 ± 1332i
    Dycal                                43000 ± 2326c                          22000 ± 5740e                         30000 ± 1868l
    ProRoot MTA                          475000 ± 53675a                        465000 ± 55629d                       333000 ± 33157h
    MTA-Angelus                          468000 ± 72158a                        522000 ± 56089d                       333000 ± 59216h
    Biodentine                           533000 ± 60897a                        592000 ± 20182d                       533000 ± 42179f


    Annali di Stomatologia 2014; V (2): 69-76                                                                                          73
    C. Poggio et al.


    tistically different from the amount of cells present in            conclusions of others Authors (39, 40) on the non-
    contact with Biodentine, ProRoot MTA and the nega-                  complete biocompatibility of calcium hydroxide-based
    tive control (P> 0001). The lowest values after 24 h is             materials: the protective effect of these materials to-
    provided by Dycal and the positive control.                         wards the pulp is not complete. Calcium hydroxide
    After 48 hours no statistically significant difference is           has an important action in protecting the pulp from




                                                                                                                                  li
    demonstrated between MTA-Angelus, Biodentine,                       thermal, mechanical and microbiological stimuli (5, 6)
    ProRoot MTA and the negative control (P> 0.001). No                 because of its antibacterial action and its property of




                                                                            na
    statistically significant difference is also present be-            stimulating sclerotic an reparative dentin formation. In
    tween Dycal and the positive control (P> 0.001),                    clinical practice, the presence of hard tissue barrier af-
    where the number of live cells is significantly lower               ter capping can be considered an asset, since it pro-
    compared to the samples of the remaining materials                  vides natural protection against the infiltration of bac-




                                                                          io
    (P <0.001).                                                         teria and chemical products. However, the importance
    After 72 hours Biodentine shows a significantly                     of calcified hard tissue barrier formation after capping
    greater number of viable cells compared to all other                has been challenged by other studies, which have
    materials tested (P <0.001), MTA-Angelus and Pro-                   shown multiple tunnel defects and cell inclusions in




                                                                       az
    Root MTA show values not significantly different be-                bridges following pulp capping with calcium hydroxide
    tween them (P> 0.001) and the negative control has                  (41). This may lead to leakage and bacteria penetra-
    retained intermediate values. Finally in the positive               tion into pulp tissue unlike the permanent seal pro-
    control samples the lower number of cells was found                 duced by bonding agents. Furthermore it is equally




                                                                    rn
    (P <0.001).                                                         demonstrated that, due to the alkalinity of its pH, calci-
    Table 3 shows the values obtained with the MTT test.                um hydroxide induces cytotoxicity, causing the forma-
    There are no statistically significant differences                  tion of a layer of coagulation necrosis, when it is in di-




                                                                 te
    among ProRoot MTA, MTA-Angelus, Biodentine and                      rect contact with the dental pulp (7). For both these
    the negative control (P>0.001). Dycal shows signifi-                reasons calcium hydroxide do not seem the eligible
    cant lower values (P<0.001).                                        material to be used in case of exposed pulp tissue.
                                                                        Very different results were obtained from the analysis

    Discussion and conclusion
                                                              In        of the MTA-based materials (ProRoot MTA and MTA-
                                                                        Angelus). Both materials to the evaluation of the 72
                                                                        hours, with Alamar blue test and MTT assay, have re-
    Pulp capping materials should act as a barrier which                ported excellent percentage of vitality, detected in a
    protects the vitality of the entire pulp tissue by cover-           range that goes from 71% to 95% of vitality and in
                                               ni
    ing the minimal exposed tissue and by preventing                    some samples the results were even assimilated to
    from further endodontic treatments. Due to this fact                the negative control. This significant difference be-
    the material used should provide an appropriate host                tween the values of vitality of calcium hydroxide and
    response; this means that tissues that come into con-               MTA is clearly due to the structural difference of the
                               o

    tact with the materials do not show any toxic, irritat-             two basic components and due to the various physio-
    ing, inflammatory, allergic, genotoxic, or carcinogenic             logical and biochemical reactions induced on tissues.
                           izi


    action (38).                                                        It has been demonstrated that MTA has the ability to
    In the present study Dycal demonstrates the lower                   induce the formation of a bridge of hard tissue of
    rates of vitality and a strong cytotoxic capability. Dycal          greater thickness compared to the bridge established
    has shown the lowest mean number of cells in the col-               in presence of calcium hydroxide, also managing to
            Ed




    orimetric assay performed with Alamar blue, with as-                cause less inflammation in the adjacent tissues (18,
    sessments at 24, 48 and 72 hours, and in the MTT as-                20). The dental pulp also contains progenitor cells
    say at 72 hours. The low percentage of vitality of Dy-              and stem cells, which can proliferate and differentiate
    cal occurs already in the first 24 hours, manifesting               into odontoblasts forming dentin; Guven and Cehreli
    small variations of 1-2 percentage points to the vari-              (42) reported that, probably, MTA is able to facilitate
    ous measurement intervals. These results confirm the                these cellular changes by inducing the secretion of
    C




                                                                        morphogenetic proteins and growth factors such as
                                                                        BMP-2 and TGF-β1.
CI




                                                                        In the present study Biodentine proved to be the
    Table 3. Mean ± standard deviation of Bonferroni post-hoc           more biocompatible material. Biodentine, in measure-
    test of the different values of cell viability. Different super-    ments made at 24, 48 and 72 hours, reported per-
    script letters indicate a statistically significant difference (P
                                                                        centage of vitality above the negative control. After
    <0.001). The same superscript letter indicates a not statis-
                                                                        24 hours it recorded values equal to 106%, rose to
    tically significant difference (P> 0.001).
                                                                        113% and 114% in the two subsequent analysis. Bio-
©




                                                                        dentine is a new bioactive cement based on calcium
    Materials                                   MTT
                                                                        silicate for pulp capping, derivation of bioengineering,
    Negative control                            124074 ± 0a             with anti-inflammatory behavior (43), different from
    Dycal                                       45741 ± 5040b           the classic materials based on calcium silicate, such
    ProRoot MTA                                 140741 ± 30098a         as Portland cement. The technology behind the man-
    MTA-Angelus                                 179444 ± 99142a         ufacturing process of the active bio-silicate, the main
    Biodentine                                  166481 ± 59317a
                                                                        constituent of Biodentine, removes the metallic impu-

    74                                                                                      Annali di Stomatologia 2014; V (2): 69-76
    Biocompatibility of pulp capping material


    rities which are present in other cements (44). The                  8. Duda S, Dammaschke T. Die direkte Überkappung – Vo-
    setting reaction involves the hydration of tricalcium                   raussetzungen für klinische Behandlungserfolge. Endodontie.
    silicate, the production of a calcium silicate-based gel                2009;18:21-31.
    and calcium hydroxide, which in contact with phos-                   9. Hørsted-Bindslev P, Vilkinis V, Sidlauskas A. Direct pulp cap-
                                                                            ping of human pulps with a dentin bonding system or with
    phate ions, it is able to create precipitated similar to




                                                                                                                                          li
                                                                            calcium hydroxide cement. Oral Surgery Oral Medicine Oral
    hydroxyapatite.                                                         Pathology Oral Radiology & Endodontology. 2009;96:591-
    Considering the interface between dentin and Bioden-




                                                                            na
                                                                            600.
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    These observations suggest how the intertubular                     11. Almushayt A, Narayanan K, Zaki AE, George A. Dentin ma-




                                                                          io
    spread produced by Biodentine hydration lead to the                     trix protein 1 induces cytodifferentiation of dental pulp stem
    creation of a hybrid layer (46). Furthermore, histological              cells into odontoblasts. Gene Therapy. 2006;13:611-20.
    evaluations carried out on samples prepared with Bio-               12. Furey A, Hjelmhaug J, Lobner D. Toxicity of Flow Line, Du-
                                                                            rafill VS, and Dycal to dental pulp cells: effects of growth fac-
    dentine have demonstrated the ability of the material to




                                                                       az
                                                                            tors. Journal of Endodontics. 2011;36:1149-53.
    induce the differentiation of odontoblasts starting from
                                                                        13. Shen Q, Sun J, Wu J, Liu C, Chen F. An in vitro investiga-
    pulp progenitor cells, forming a mineralizing matrix with               tion of the mechanical-chemical and biological properties of
    the characteristics of dentin (47).                                     calcium phosphate/calcium silicate/bismutite cement for den-
    In conclusion Biodentine has shown to be the materi-




                                                                    rn
                                                                            tal pulp capping. Journal of Biomedical Materials Research
    al with the best qualities and characteristics, that are                Part B: Applied Biomaterials. 2010;94:141-8.
    the basis of biocompatibility. Because of the lower cy-             14. Torabinejad M, Parirokh M. Mineral trioxide aggregate: a com-
    totoxicity and the higher bio-inductive ability, Bioden-                prehensive literature review part ii: leakage and biocom-
                                                                            patibility investigations. Journal of Endodontics. 2013;36:190-




                                                                 te
    tine can be considered an ideal cement for pulp-cap-
    ping. Nevertheless, especially for the new generation                   202.
                                                                        15. Takita T, Hayashi M, Takeichi O, Ogiso B, Suzuki N, Otsu-
    materials, further studies must be started to demon-
                                                                            ka K, Ito K. Effect of mineral trioxide aggregate on prolifer-
    strate the clinical efficacy and illustrate the actual
                                                                            ation of cultured human dental pulp cells. International En-
    mechanisms of action, both in vitro and in vivo.
                                                              In            dodontic Journal. 2006;39:415-22.
                                                                        16. Sawicki L, Pameijer CH, Emerich K, Adamowicz-Klepalska B.
                                                                            Histological evaluation of mineral trioxide aggregate and cal-
    Acknowledgments                                                         cium hydroxide in direct pulp capping of human immature per-
                                                                            manent teeth. American Journal of Dentistry. 2008;21:262-6.
                                                ni
    Nothing to declare.                                                 17. Gandolfi MG, Shah SN, Feng R, Prati C, Akintoye SO. Bio-
                                                                            mimetic calcium-silicate cements support differentiation of
                                                                            human orofacial bone marrow stromal cells. Journal of En-
    Conflict of interest statement                                          dodontics. 2011;37:1102-8.
                               o

                                                                        18. Moghaddame-Jafari S, Mantellini MG, Botero TM, McDon-
                                                                            ald NJ, No¨r JE. Effect of ProRoot MTA on pulp cell apop-
    The authors of this study have no conflict of interest
                           izi


                                                                            tosis and proliferation in vitro. Journal of Endodontics.
    to disclose.                                                            2005;31:387-91.
                                                                        19. Okiji T, Yoshiba K. Reparative dentinogenesis induced by
                                                                            mineral trioxide aggregate: a review from the biological and
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