Karpukhina Bioactivity and Fluoride Release 2017 Accepted

Bioactivity and Fluoride Release of Strontium and &Fluoride Modified Biodentine Hazel O Simila – 1, Natalia Karpukhina – 2, Robert G Hill – Department of Conservative and Prosthetic Dentistry, University of Nairobi, Kenya, Dental Physical Sciences Unit, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, UK Introduction Biomaterials science aims to develop materials that are ideal mechanically, physically and biologically Previously, most research effort was directed at the physico-mechanical properties of materials with less attention to biological properties However, recently that focus seems to be shifting towards developing the bioactive aspect of biomaterials due to interest in minimally invasive procedures [1] Bioactivity is the induction of a favourable host response by a foreign implanted material leading to interfacial bond formation with biological tissues This could be due to an effect on cells, hormones or other chemical signalling factors such as plasma proteins, the response thereof leading to reversal of disease process A bioactive dental material should promote deposition of calcium hydroxyapatite crystals which promotes bonding with the remnant dental tissues [2] Some bioactive dental materials include calcium hydroxide cements, glass ionomer cement, mineral trioxide aggregate (MTA), and other newer tricalcium silicate (TCS) based cements such as BiodentineTM TCS based cements form a substantive mineral infiltration zone at their interface with the tooth and stimulate transforming growth factor (TGF) β1 production which is needed in inducing progenitor cell differentiation and dentine tissue deposition [3,4] Therefore, despite these materials having inferior mechanical properties, they can reverse disease progression through multiple mechanisms [5] These cements are superior to either calcium hydroxide or glass ionomer cements due to the extent of mineral uptake and infiltration [6,7]; superior sealing ability [8]; pronounced effect on TGF β1[4]; speed with which they form a mineralized layer [9]; and the minimal inflammation that accompanies the process [10] MTA, the prototype TCS cement was first discovered at Loma Linda University [11,12] Although having desirable biological properties, MTA is a difficult cement to manipulate due to its grainy consistency and long setting time [13] These limitations led to Septodont developing BiodentineTM, using active Biosilicate technology [14] BiodentineTM sets in approximately 12 minutes and is effective in pulp therapy, while being able to act as a coronal dentine replacement This cement is presented as a powder and liquid The powder contains; tri-calcium silicate (C3S), di-calcium silicate (C2S), calcium carbonate filler, iron oxide shade, and zirconium oxide radiopacifier while the liquid has a calcium chloride accelerator and a hydrosoluble polymer [15] Generally TCS cements promote formation of a thick dentine layer by initial production of calcium hydroxide cement during setting [6] and by ion infiltration into dentine [16] BiodentineTM also induces pulp cell differentiation into odontoblast like cells that facilitate tertiary dentine formation It also increases alkaline phosphatase activity and secretion of TGF β1 [4]which is responsible for odontoblast differentiation during tooth morphogenesis [17] Despite BiodentineTM’s desirable properties, there is room for improvement by incorporation of the caries inhibiting fluoride and strontium species Fluoride confers anticariogenic properties due to inhibition of plaque bacteria metabolism; impairing plaque bacteria adhesion and build up; and formation of acid resistant fluorapatite crystals [18] Additionally, fluoride has a significant buffering effect that drives remineralization [19] This caries inhibiting effect has led to extensive effort to incorporate and evaluate the fluoride release of various restorative materials [20] Strontium on the other hand is a species that has been shown to manage dentine hypersensitivity [21] and lead to caries inhibition [22] Moreover, its synergistic effect with fluoride in caries prevention is well documented by Lippert and Hara [23,24] who summarize earlier research by Curzon that demonstrated the caries inhibiting capability of strontium [25,26] Bioactive glass can be used as a carrier for mineral species due to its dissolution leading to release of ions and independent apatite forming ability This dissolution is dependent on the network connectivity of the bioactive glass, which in turn is influenced by its composition, thus, can be controlled[27] The first bioactive material bearing the Bioglass trademark was first discovered by Larry Hench between 1969 and 1971 [28,29] This material forms apatite when in the bio-physiological environment and has been utilized in modifying other materials for dental applications[30,31] Their apatite forming ability has been employed in nouvelle approaches to management of dentine hypersensitivity whereby apatite formed is useful in occluding open dentinal tubules [32,33] The aim of this study was to incorporate fluoride and strontium containing bioactive glasses into BiodentineTM and assess the impact on apatite formation and ion release of the modified cement The specific objectives were to; (i) Synthesize three types of bioactive glasses; create three versions of modified BiodentineTM cements in addition to unmodified cements by adding 10% by weight of high fluoride bioactive glass; 10% high strontium bioactive glass; or 10% fluoride plus strontium bioactive glass into BiodentineTM powder before mixing with (ii) BiodentineTM fluid Use FTIR, XRD and NMR to characterize the structure of all the cements after (iii) immersion in PBS for and 24 hours, 3, and 14 days Determine the fluoride ion release profile of the two cements modified with fluoride containing bioactive glass Materials and methods The bioactive glasses were prepared using a melt quench route Mixtures of analytical grade SiO (Prince Minerals Ltd, Stoke-on-Trent, UK), P2O5, CaCO3, Na2CO3 and CaF2 (all Sigma-Aldrich, Gillingham, UK) were prepared according to the compositions in (Table 2) The weight percentages were correlated to the molar weight to maintain a desirable degree of network connectivity, important for bioactivity Each composition was melted in a platinum–rhodium crucible for h at 1430 °C in an electric furnace (EHF 17/3, Lenton, Hope Valley, UK) After melting, the glasses were quenched in water to prevent crystallization and the frit retrieved using a large sieve The frit was dried overnight in an oven at 50°C Later, the glass was ground using a Gyro-mill (Glen Creston, Wembley, London, UK) for minutes and thereafter sieved using a 90 μm mesh analytical sieve XRD confirmed the amorphous nature of the two bioactive glass compositions, however, the third glass (F- + Sr = H) partially crystallized to strontium fluoride SrF2 The XRD patterns are provided in the supporting material (Figure S1) Table 1: Compositions of the bioactive glasses in mol% Bioactive glass SiO2 P2O5 High F- - Q 36.8 0.8 High Sr – I 38.1 F- + Sr – H 36.8 SrO Na2O CaO CaF2 SrF2 - 19.6 17.2 25.5 - 6.3 25.9 29.6 - - - 0.8 17.2 19.6 - - 25.5 2.1 Cement preparation Four different types of cements were manipulated Plain BiodentineTM cement coded ‘BO’, was prepared by adding five drops of fluid to the powder and triturating for 30 seconds in a 3000rpm electric amalgamator (3M Insert equipment detailsESPE RotoMix, UK) All modified BiodentineTM samples were prepared by adding 0.07g of the specific bioactive glass into the BiodentineTM containing capsule This amount was approximately 10% of the BiodentineTM content The first modification involved addition of 0.07g of high F- (Q) bioactive glass into a BiodentineTM powder containing capsule This was mixed in the 3000rpm amalgamator used for BiodentineTM manipulation for 10 seconds To this mix, five drops of BiodentineTM fluid were added and amalgamated for 30 seconds to achieve a creamy paste This cement sample was coded ‘BQ' Similar steps were followed to produce cement sample ‘BI’ and ‘BH’, using high Sr (I) bioactive glass and high F- + Sr (H) bioactive glass respectively All three modified cements for testing contained bioactive glass: BiodentineTM in the ratio of 1:10 The unmodified BiodentineTM cement acted as a control 2.2 Specimen sample preparation Cylindrical specimens from all the four cements measuring 4mm diameter and 6mm high were prepared from steel moulds These were allowed to set for 30min in a humid water bath maintained at 37oC After setting, a single cylindrical sample was immersed in 10ml of phosphate buffered saline (PBS) in a 25ml tube This was kept in an orbital shaker ( IKA KS 4000i, GermanyInsert equipment details) maintained at 37 O C at an agitation rate of 60 rpm For each cement, such specimens were prepared, labelled and stored for and 24 hours, 3, and 14 days At the expiry of each immersion period, the samples were retrieved, and the solutions were filtered through medium porosity filter paper (5 µm particle retention, VWR International, Lutterworth, UK) The solutions from the BQ and BH cements were stored at 4oC for later fluoride ion release studies Filtered powder, plus the filter paper was dried overnight in a 37 oC oven The powder was retrieved and ground together with the original cylindrical specimen that had been immersed in the PBS solution The powders were appropriately labelled based on composition and immersion period and characterized 2.3 Characterization of the powdered samples Commercial hydroxyapatite powder (Plasma Biotal Ltd.) and the four cement powders were analysed using Fourier Transform Infra-Red (FTIR) spectroscopy (Spectrum GX, Perkin Elmer, UK)Spectrum GX, Waltham, MA,USA; and data collected from 400 cm-1 and 4000 cm-1) X-ray diffraction (XRD) analysis on an XRD machine (X’Pert PRO MPD, PANalytical, Cambridge UK) was also performed on the cement powders using a D5000 diffractometer (Phillips PW1700, 40kV, 40mA of , CuKα X-rays) The data was collected at room temperature with a 0.04o 2 step and a count rate of s per step, from 2 values of - 70 degrees X-ray diffraction patterns obtained were matched with reference values taken from Powder Diffraction Data File (PDF) of the International Centre for Diffraction Data (ICDD) Based on FTIR and XRD results, structural analysis of specific sample powders using 19F and 31P magic angle spinning nuclear magnetic resonance (MAS NMR) was performed using a Bruker 600MHz (14.1 T) spectrometer (AV600, ( Insert equipment detailsBruker, Germany) A resonance frequency for 31P nucleus was determined at 242.9 MHz A given sample powder was packed into the 4mm diameter rotor made of zirconium oxide at any one given test time Spinning frequency was set at 10 kHz Topspin software was used for setting the parameters, running NMR experiments and for viewing and plotting the NMR spectra Calibration of the chemical shift scale was done prior to commencing the experiment using the signal from the 85% H3PO4 solution assigned to ppm Recycle delay was 60s and the total number of scans done was in multiples of All spectra were recorded at ambient probe temperatures Only the 14 day BQ cement was subjected to the 19F MAS NMR spectroscopy The spectrum was run at the 564.7MHz using a low fluorine background Bruker probe in a 2.5 mm rotor spun at 20 kHz with 30s delay for the cements The 19F chemical shift scale was referenced using 1M NaF solution giving a signal at -120 ppm against CF3Cl 2.4 Fluoride ion release studies Fluoride ion release studies were done on the BH and BQ cements These two cements were selected since they we modified with fluoride containing bioactive glasses BH was modified with fluoride + strontium bioactive glass, while BH was modified with fluoride only bioactive glass Fluoride release into the PBS solution was measured using a fluoride ion selective electrode (Orion 9609 BNWP with Orion pH/ISE metre 710, Thermo Scientific) Measurements were taken twice Calibration of the fluoride electrode was done using PBS solutions of 100ppm, 50 ppm, 25ppm, 10 ppm, 1ppm and 0.5ppm 5mg/L to account for ionic strength Using the equation of the line from the calibration, the mg/L (ppm) values were calculated and plotted against time Results From the FTIR analysis shown in Error: Reference source not found, Plasma Biotal yielded main apatite peak at 1025 cm-1 and prominent split peaks in the 560 cm-1 - 600 cm-1 range The BO, BQ, BI and BH samples were compared against this Overall, these cements show signal at 1025 cm-1 and split peaks at 560cm-1, 600 cm-1 These peaks are consistent with apatite formation, with the split peak also potentially representing a crystalline precursor of apatite, such as octacalcium phosphate However, when compared to the reference apatite peaks of Plasma Biotal, the 1025 cm-1 peak is less pronounced in most of the experimental cements except for BQ at 14 days The intensity of the split peaks at 560cm-1, 600 cm-1 increased with increasing immersion time for all cements There are other observable peaks in the 1440cm-1 – 1460cm-1 range and at 870 cm-1 position which show the presence of carbonate in apatite The intensity of these peaks seems prominent in the BO cement, which is also consistent with presence of calcium carbonate in the unmodified cement In Figure 2Error: Reference source not found below, a comparison profile of the cements’ XRD patterns after day immersion in PBS is presented These XRD patterns show presence of tricalcium silicate (C3S), dicalcium silicate (C2S), calcium carbonate (CaCO3) and calcium hydroxide (Ca(OH)2) as the main crystalline phases Other than an intenisty change in the marked calcium hydroxide diffraction line , the rest of the patterns are almost identical Figure 3a demonstrated 31P MAS NMR spectra for the cements after hours of immersion All the spectra clearly reveal presence of a relatively sharp signal at about ppm, which is near the position of the signal in crystalline hydroxyapatite as seen from the top spectrum The 31P spectrum of the BI sample showed an additional broad feature at about ppm It can be seen from the Figure 3b that this feature belongs to the original glass, which remains a significant fraction at hours, though largely disappears after 24 hours as seen from the Figure 3b The 19F MAS NMR spectrum for BQ at 14 days is shown in Figure along with the spectrum of the original glass before it was added to the cement There is a relatively broad peak at about -103.2ppm that appears on the immersion and was not present in the original glass This is assigned to the fluoride-substituted apatite Some fluoride signal that belongs to the bioactive glass environment is seen in addition to the fluorapatite in the spectrum The results of the fluoride ion concentration measurements in solutions given in Figure indicate low fluoride release, which is proportional to the low amount of fluoride incorporated into the cements BH seems to have a higher release compared to BQ despite having a lower fraction of F- The release seems to be increasing over the time points Discussion Plain BiodentineTM forms apatite when immersed in PBS, as seen in the FTIR spectra (Figure 1) and this has also been reported previously [34] From the 31P MAS NMR it was seen that the apatite fraction of the modified cements was higher at the same immersion period compared to the plain BiodentineTM Figure 3a reveals slight variation in the line width of this signal between the samples which is likely due to a different crystalline size Certainly these new apatite crystals forms are smaller than the commercial powder This is seen from the comparison of the signalto-noise ratio Poor signal-to-noise ratio seen especially from the BOfor some spectraum is due to a low amount of apatite phase formed at hours This is because For instance, in the plain Biodentine, PBS the immersion media was the only source of phosphorus was PBS solution, and which means apatite resulted from the precipitation from the solutionthe phosphate signal observed was due to a precipitation of apatite-like phase The other spectra show better signal-tonoise ratio; there was additional phosphate source from the bioactive glass additives Formation of fluorapatite in place of hydroxyapatite is also demonstrated The 19F MAS NMR of BQ confirms presence of fluorapatite as a result of the re-mineralizing reaction in PBS The relatively broad signal at -103.2ppm is an indication of small crystal size of the fluorapatite (Figure 4) Although BH was not assessed by 19F MAS NMR, it is fair to predict that the apatite observed through FTIR for this specimen (Figure 1), would be fluorapatite too Presence of fluorapatite in BQ is consistent with the low release of fluoride observed from the fluoride ion release findings of Figure Generally, ion release is expected to correlate to compositional amount The higher the initial amount, the higher the release expected The anticipated fluoride release for BQ and for BH cements is approximately 261mg/L and 197mg/L respectively but this is estimated taking into account all fluoride present, regardless of whether any fluoride is tied up in fluorapatite The measured release starting at days is at 1% of this total amount and from this; we can speculate that the long termlong-term release at measured ppm should be beneficial The higher fluoride release seen in BH cements (Figure 5) may be due to the presence of strontium which promotes release of other ions The BH cement was also shown to form apatite at earlier time points of hours and this can be explained by the effect of strontium on calcium ion release [35] Although strontium does not grossly affect chemical structure when substituted for calcium in bioactive glasses, it tends to weaken the chemical structure and enhance dissolution [36] Strontium cation is slightly bigger than calcium cation and therefore an expansion of silicate matrix can be expected in case of strontium substitution This can cause earlier glass dissolution compared to the calcium only bioactive glass [37] While BH cement may have benefited from presence of strontium, BI formulation additionally contained a bioactive glass with the highest phosphate content (Table 1), which is an influential element known to form apatite much earlier in buffer solutions [31] However, the BI formulation cements showed a large fraction of the orthophosphate signal (Figure 3) indicating significant presence of the remaining glass phase unlike other formulations This is explained by the high phosphate content in the bioactive glass used in this formulation BI formulation had a glass with 6.3mol% of P2O5 compared to the other two glass containing formulations with P2O5 content below 1mol% A combination of reasons can explain this The dissolution of the phosphate from the glass might be suppressed in phosphate rich solutions like PBS compared to the low phosphate glasses Secondly, the BI formulation has no fluoride in its composition and finally, suspected bigger particle size of the glass powder used for BI would potentially slow down the glass dissolution as well BQ was shown to have the most prominent apatite peak at 14 days (Figure 1) Being the cement with the high fluoride containing bioactive glass, it is expected that the high fluoride content causes a reduced pH rise, favoring formation of fluorapatite It is thought that the stability of the fluorapatite formed allows accumulation of apatite and hence cancels out the effect of lower pH associated with lower apatite formation [38] Although BH had fluoride too, it is likely that the crystallization of the strontium fluoride phase affected the bioactive glass dissolution leading to less prominent apatite formation It is important to note that despite this fluorapatite formation behavior, the fluoride release profile of BH was higher than that of BQ (Figure 5) It is possible that since BQ demonstrated the highest fluorapatite formation (Figure 1), this could mean depletion of the fluoride which could significantly lower the fluoride release of this cement Hence, strontium fluoride present in BH would not affect fluoride release as much as fluorapatite formed by the BQ cement These observations are expected given the earlier discussion on the influence of strontium on apatite and ion release profile of BH It is worth noting that although apatite formation was shown via FTIR (Figure 1) and 31P MAS NMR 9(Figure 3a & 3b3); the same could not be said of XRD Although a previous study of BiodentineTM found some broad diffraction lines at the 320-340 2ϴ range attributed to apatite formation, no such observation or conclusion is made in this study [39] It is possible that the apatite present was too little or nanocrystalline to be demonstrated by XRD (Figure 2) Moreover, BiodentineTM is a multiphase cement, with prominent silicate peaks in the same range that would overshadow any apatite presence However, XRD was still helpful in revealing the calcium hydroxide peaks at 18.0 2ϴ degrees Although no conclusive remarks can be made regarding the prominence of this peak, it is important to note that calcium hydroxide is a reaction product in the setting of any tricalcium silicate cement [40] Calcium hydroxide presence indicates an excessive calcium release on immersion of calcium silicates which provides the calcium ions needed to form apatite The immersion study here was doneduring immersion in PBS in order to provide the media with a phosphate, which is also needed for apatite formation without adding additional calcium concentrations the calcium ions needed to form apatite From XRD, it was established that at day (Figure 2), days and 7days immersion, BI, BQ and BO had the most prominent calcium hydroxide peaks, respectively By 14 days, the proportion of this phase was nearly the same in all cements This study provides an indication of the possible biological sequelae of incorporating bioactive glass into BiodentineTM Proof of fluorapatite formation via MAS NMR is consistent with interpretation of data from FTIR spectra XRD patterns are not conclusive with regard to apatite formation but yield equally important information on calcium hydroxide formation This information is of prime importance in stirring effort to modify and improve BiodentineTM Acknowledgements This research was presented at the 2014 International Association of Dental Research conference in Cape Town, South Africa It was based on a thesis submitted to the graduate faculty, Queen Mary University of London, in partial fulfilment of the requirements for the MSc Dental 10 Materials degree Sponsorship for the graduate study was graciously offered by the University of Nairobi The authors declare no potential conflict of interest with regard to authorship/publication of this article We would like to specially recognize and thank Dr Rory Wilson for his tremendous help with the XRD characterization Similarly, Dr Andy Bushby offered significant direction as course convener References [1] Bayne SC Dental Biomaterials: Where Are We and Where Are We Going? 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(b) 31P MAS NMR spectra of the BI experimental cements samples plotted at different immersion 16 times The bottom spectrum is for the untreated bioactive glass used as an additive in the cement The top spectrum in (a) and (b) is for the commercial hydroxyapatite used as a reference Figure 4: 19F MAS NMR spectra of the Biodentine modified with high F bioactive glass (BQ) after 14 days immersion in PBS Figure 5: Fluoride ion release of BH and BQ cements 17 ... ppm against CF3Cl 2.4 Fluoride ion release studies Fluoride ion release studies were done on the BH and BQ cements These two cements were selected since they we modified with fluoride containing... higher the release expected The anticipated fluoride release for BQ and for BH cements is approximately 261mg/L and 197mg/L respectively but this is estimated taking into account all fluoride present,... could mean depletion of the fluoride which could significantly lower the fluoride release of this cement Hence, strontium fluoride present in BH would not affect fluoride release as much as fluorapatite