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Journal of Alloys and Compounds

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Introducing the fluorine doped natural hydroxyapatite-titaniananobiocomposite ceramic

Ebrahim Karamian a, Majid Abdellahi a, *, Amirsalar Khandan b, Sana Abdellah a

a Advanced Materials Research Center, Faculty of Materials Engineering, Najafabad Branch, Islamic Azad University, Najafabad, Iranb Young Researchers and Elite Club, Khomeinishahr Branch, Islamic Azad University, Isfahan, Iran

a r t i c l e i n f o

Article history:Received 2 December 2015Received in revised form24 February 2016Accepted 7 April 2016Available online 10 April 2016

Keywords:FNHA-TiO2 nanobiocompositeHydroxyapatiteBioactivity

* Corresponding author.E-mail address: M.Abdellahi@Pa.iut.ac.ir (M. Abde

http://dx.doi.org/10.1016/j.jallcom.2016.04.0680925-8388/© 2016 Elsevier B.V. All rights reserved.

a b s t r a c t

In the present research, natural hydroxyapatite (NHA) was synthesized from bovine bones and thenfluorine was doped into the NHA matrix to produce fluorine doped NHA (FNHA; natural fluor-hydroxyapatite) in optimum conditions. At the end an FNHA-TiO2 nanobiocomposite ceramic withexcellent biocompatibility and good chemical stability was synthesized through a mechanochemicalroute and a subsequent two step sintering (TSS) process. Thermal gravimetric analysis (TGA), Differentialscanning calorimetry (DSC), X-ray fluorescence (XRF), X-ray diffraction (XRD), scanning electron mi-croscopy (SEM), inductive coupled plasma (ICP), and energy-dispersive X-ray spectroscopy (EDX) wereused as the means for gathering and analysis of the results. According to the obtained results, TiO2 canprevent early decomposition of FNHA by the formation of the CaTiO3 phases and hence strengthen theinteractions between the apatite particles which results in the increase of the mechanical properties.Besides, TiO2 provides more SieOH nucleation sites for the formation of the apatite layers and hencemore bioactivity.

© 2016 Elsevier B.V. All rights reserved.

1. Introduction

Hydroxyapatite (HA) is a naturally occurring mineral form ofcalcium apatite with the formula Ca5 (PO4)3(OH). It is usuallywritten as Ca10 (PO4)6(OH)2 with the Ca/P ratio being 1.67. HA is thehydroxyl end member of the complex apatite group [1]. Amongdifferent calcium phosphate ceramics, HA has been extensivelyused as a substitute material for bone or damaged teeth because ofits crystallographic similarity to various calcified tissues of verte-brates [2]. However, HA is faced with an intrinsically high disso-lution rate in a biological environment and poor corrosionresistance in acid solutions [2]. Moreover, HA has a poor thermalstability which results in its decomposition into other phases andhence undesirable fast dissolution rate in vivo [3].

Fluorine doped hydroxyapatite (F-HA; Ca10 (PO4)6(OH)2�xFx hasbeen revealed to be a viable alternative to bone because of its goodbiocompatibility, low solubility, and also high thermal and chemi-cal stability [4]. Pure fluorapatite (FA; Ca10 (PO4)6 F2) is known tohave a much lower solubility in biological fluids than HA, because

llahi).

FA has a greater stability compared to HA, both chemically andstructurally. According to a study conducted by Eslami et al. [5],with increasing the incorporation of F into the HA structure thermalstability is considerably increased. By varying the amount of Fluo-ride substitutions the solubility as well as biological lifetime can befine-tuned. The results of cell culture demonstrated that the Fcontent affects the cultured cells behaviours in different ways: ahigh F content leads to a low surface potential, which favours cellattachment. However, a decrease of the Ca2P release could inhibitcell proliferation. Fluorine can also act as a superior substitute tothe coating of titanium implants. Tredwin et al. [6] showed thatwith increasing the F content at all calcination temperatures,especially at 800 and 1000 �C, the bond strength can be increased.

It should also be stated that with high concentrations of fluoridein groundwater, two most critical issues arise: dental and bonefluorosis which can evolve into a more severe illness such asosteosclerosis or exostosis [4]. Accordingly, it is necessary to selectan optimum content of F� in FHA in order to optimize the bioac-tivity of the implants to control the amount of fluoride ions releasedfrom FHA [4]. Besides, themechanical properties of fluorapatite andall other calcium phosphates are generally inadequate for manyload-carrying applications [7]. These bioceramics also have a lowdensity and hence poor mechanical properties [7]. Research works

Delta:1_given nameDelta:1_surnameDelta:1_given nameDelta:1_surnamemailto:M.Abdellahi@Pa.iut.ac.irhttp://crossmark.crossref.org/dialog/?doi=10.1016/j.jallcom.2016.04.068&domain=pdfwww.sciencedirect.com/science/journal/09258388http://www.elsevier.com/locate/jalcomhttp://dx.doi.org/10.1016/j.jallcom.2016.04.068http://dx.doi.org/10.1016/j.jallcom.2016.04.068http://dx.doi.org/10.1016/j.jallcom.2016.04.068

Table 1XRF analysis of the hydroxyapatite powders.

Element Ca P Na F Mg Sr Cl Si S Al Cu Zn Fe K Zr Ca/P Total

wt% 70.2 21.09 1.14 1.09 0.64 0.40 0.18 0.12 0.06 0.05 0.05 0.04 0.04 0.02 0.007 3.33 100.04

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carried out in this relation show that the combination of calciumphosphates and other compounds can improve the poor mechan-ical properties of calcium phosphates [7].

Some attempts have been made to develop the HA-basedcomposites such as the HA-Al2O3 [8], HA-ZrO2 [9], and HA-TiO2[10] composites. These studies point to the occurrence of twomajorcommon phenomena including the dissociation of HA to tricalciumphosphate (TCP) and interfacial reactions between HA and rein-forcement ceramic phasewhich can lead to the formation of CaZrO3and CaTiO3 in HA-ZrO2 and HA-TiO2 systems, respectively. How-ever, only a few studies have concentrated on the use of metallicoxides such as TiO2 for the preparation of the FA-based bio-nanocomposites [11]. Therefore, the main objective of the presentresearch is the fabrication of FNHA-TiO2 biocomposite which canpresent the advantages of both TiO2 and FNHA.

HAeTiO2 nanobiocomposite is one of the most importantachievements of the mentioned researches. It is produced bydifferent methods such as chemical co-precipitation [12], in situprecipitation [13], photo-induced formation [14], sol-gel dipcoating [15], and gas tunnel type plasma spraying [16]. In additionto the above-mentioned methods, the high energy ball millingprocess is a simple dry method to obtain any quantity of powderwith controlled microstructure [17]. Usually, the powder obtainedby the mechanochemical route has a suitable structure because ofthe disorderliness of surface-bonded species resulted by pressure[18]. The benefits of ease, reprocessing capability, and low pro-cessing cost are the most significant advantages of this method[19]. Moreover, it is not necessary to precisely control the meltingconditions and the obtained powders have nanostructural charac-teristics [20].

Despite the poor mechanical properties of hydroxyapatite andits compounds, their unique biological properties lead us to thinkabout working on improving their properties instead of completelyreplacing them by other materials. In the present study, at first theHA powders were produced in natural form through a very simplemethod. To obtain better chemical stability, a controlled amount offluorine was injected into the hydroxyapatite matrix. Finally, anFNHA-TiO2 bioceramic composite with an optimum amount of TiO2was fabricated and its chemical stability and biological propertywere assessed. The obtained results promised a nanobiocompositewith good chemical stability and biological properties.

2. Materials and methods

2.1. Preparation of the powder samples

As a brief reiteration of the methods in our previous work [21],

Table 2Ion concentrations (mmol/dm3) of SBF and Human Blood Plasma.

Ionic concentration (mmol/dm3) Simulated body fluid Blood plasma

Naþ 142.0 142.0Kþ 5.0 5.0Mgþ2 1.5 1.5Caþ2 2.5 2.5Cl� 147.8 103.0HCO�3 4.2 27.0HPO4�2 1 1.0SO4�2 0.5 0.5pH 7.4 7.2e7.4

bovine bones were boiled for 12 h to remove their fat. In order toremove moisture, the wet bones were heated to 110 �C and kept atthis temperature for 2 h. To prevent the blackening with sootduring heating, the bones were cut into approximately 10-mmthick pieces and heated to 550 �C for 2 h in air to allow evaporationof organic substances. The blackened bone pieces were heat-treated for 3 h at 850 �C. The powdered mixture was then loadedinto a hardened steel bowl along with a ceramic ball with 10 mmdiameter and 110 gr mass. The ball-to-powder weight ratio (BPR)was 15, and the rotational speed was 600 rpm. The sample wassubsequently heated in a furnace to 850 �C for 8 and 10 h. The timeof milling by using the planetary ball mill was set on 10 h. The XRFanalysis which confirms a successful synthesis of HA is presented inTable 1. It is worth mentioning that the amount of Ca/P in theproduced HA (natural hydroxyapatite) is more than that in the non-natural one (Ca/PHA ¼ 1.67 and Ca/PNHA ¼ 3.33).

2.2. Synthesis of FNHA

The natural hydroxyapatite (NHA) powders obtained from theabovementioned process were again ball milled for 0.5 h to ho-mogenize the product. The FNHA particles were produced byblending NHA and CaF2 by a high energy planetary mill. The BPR of15 and rotational speed of 600 rpm were used for the millingprocess in which the overall mass was 135 g and milling durationswere 4, 6, and 8 h.

2.3. The composite formation

An FNHA-TiO2 nanobiocomposite powder with different per-centages of TiO2 (5 wt%, 10 wt%, and 15 wt%) was mechanicallyactivated through high energy ball milling. The BPR was 10 and thespeed of vial was 600 rpm. The powders were weighed in amountsof 0.3 g and pressed in a 6 mm diameter steel disc mould for 2 minunder a force of 9000 N. Thereafter, the mixed powders were coldpressed under 600 MPa, and the prepared compact samples weresintered through a two-step sintering (TSS) process (T1 ¼ 1150 �C,T2 ¼ 950 �C). The sintered specimens were cooled slowly in afurnace at the cooling rate of 10 �C/min (The TSS diagram was sentto the Journal office as a supplementary file).

2.4. The characterization procedure

Phase and structural analyses were carried out by using XRD(Philips, X'Pert MPD diffracto-meter) using CuKa radiation(l ¼ 0.15418 nm) over a 2q range of 20e70�. The achieved experi-mental patterns were compared with the standard patternscompiled by the Joint Committee on Powder Diffraction and Stan-dards (JCDPS), represented by card no. 09-432 for NHA. Scanningelectron microscopy (SEM) (Philips XL30, Eindhoven, TheNetherlands) was used to observe the morphology of the surface.The Ca/P ratio was determined by using energy-dispersive X-ray(EDX) spectroscopic microanalysis. The samples were coated withAu for 2 min by spraying in high vacuum at the accelerating voltageof 35 kV to be suitable for SEM investigations. The concentrations ofCa, Si, … ions in Simulated Body Fluid (SBF) after soaking weretested using inductive coupled plasma atomic emission spectros-copy (ICP-AES; Zaies 110394c). The elemental analysis of hy-droxyapatite powder (raw material) was performed by X-ray

Fig. 1. Two-step sintering (TSS) process used in this work.

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fluorescence spectrometry (XRF, Bruker-S4 Pioneer, Germany). TheDSC experiments were performed on a differential scanning calo-rimeter (DSC 60, Shimadzu Co., Japan). The samples were accu-rately weighed in aluminium pans and heated at 10 �C/min under anitrogen flow. Thermal gravimetric analysis (TGA) of the as-prepared powders was carried out on a Mettler-Toledo TGA 851e.

2.5. In vitro bioactivity evaluation

The SBF (Simulated Body Fluid) test is a technique that is used toevaluate the in vitro bioactivity of materials [22]. In this method,the selected materials are immersed in an aqueous SBF solution inwhich the characteristics of human blood plasma have beensimulated. After immersion for a certain period, the bioactivity isevaluated by the amount of the HA layer formed on the surface ofthe samples [23]. The SBF solution is produced in laboratory withthe ionic concentration nearly similar to human blood plasma, ac-cording to the procedure proposed by Kokubo (known as Kokubomethod) [24]. The appropriate quantities of reagents comprised ofNaCl, NaHCO3, KCl, K2HPO4$3H2O, MgCl2$6H2O, CaCl2, Na2SO4, andtris buffer are dissolved in 1 l of double distilled water so as to haveionic concentration of various inorganic ions similar to those of the

Fig. 2. DSC/TGA data for as prepared powder of NHA.

human blood plasma [24]. Table 2 provides information on ionsconcentration of SBF obtained by Kokubo method and its compar-ison with human blood plasma (see Fig. 1).

3. Results and discussion

The DSC/TGA data for the as-prepared NHA powder are shownin Fig. 2 in which the first endothermic peaks at 100 �C and 250 �Care related to the absorbed water. As can be seen, two exothermicpeaks were obtained between 400 �C and 600 �C without anysignificant weight loss. So, it can be claimed that they are corre-sponding to some kind of phase transformation (a-TCP (Ca3 (PO4)2)to a0 and beTCP). This is verified by Fig. 3 in which the X-ray pat-terns of the as-synthesized NHA powder and that of calcined at560 �C are presented. It is observable from this figure that bothpatterns contain HA peaks; however, with increasing the temper-ature to 560 �C, distinctions become visible in the appearance of thenew beTCP phases. Another exothermic peak, which can be seen at750 �C, probably represents DCP (Ca2P2O7). A weight loss close to21% is observed in the TGA graph. This leads to the conclusion thatNHA does not have a suitable stability in high temperatures andchemical solutions.

As it was mentioned before, NHA is able to form FNHAwith thecrystallographic substitution of OH� by F� which has a much lowersolubility than NHA, making it an alternative potential candidatefor bone repair. Fig. 4 shows the TGA graph for NHA at differentpercentages of fluorine. Several important points can be inferredfrom this figure. First, the weight loss of NHA has been markedlyreduced by adding fluorine which means an increase in chemicalstability. Second, in low percentage of fluorine, there will be athermal instability at 850 �C like pure NHAwhich means that thereis an optimum content of the fluorine doped in NHA. As can be seenin this figure, by increasing the fluorine content, the OH groups arecompletely substituted by fluorine during which FA with goodchemical and thermal stability is formed. These observations sug-gest that when the fluorine content in the NHA matrix is highenough, the thermal stability of FNHA improves and in the corre-sponding expression the decomposition of FNHA would be effec-tively postponed.

Fig. 5 indicates the XRD patterns of the NHA doped with CaF2 atvariousmilling times. The broadening of the peaks can be seenwithincreasing the milling time which results in crystallite size refine-ment [25]. It is also worth considering that FNHA is completelyformed at 8 h, which implies that the doping process has beencompleted at or before this milling time. To further explore the

Fig. 3. X-ray patterns of as synthesized NHA powder and NHA powder calcined at560 �C (* points are new phases).

Fig. 4. TGA graph for NHA at different percentage of fluorine.

Fig. 5. XRD patterns of the NHA doped with CaF2 at various milling times.

Fig. 6. The FHNA- TiO2 nanobiocomposite samples after TSS process; a) 5%wt TiO2; b)10%wt TiO2 and c) 15%wt TiO2.

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Fig. 7. X-ray diffraction pattern of sintered FNHA/TiO2 nanobiocomposite.

Fig. 8. TEM images of the sintered samples; a) 10%wt TiO2 and b) 15%wt TiO2.

Fig. 9. The changes in a) calcium ions concentration and b) pH values versus immersion time in the SBF solution.

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Fig. 10. SEM images of the FNHA- TiO2 nanobiocomposite samples after immersion in SBF solution for 21 days; a) pure FNHA; b) FNHA-5%wt TiO2; c) FNHA -10%wt TiO2 and d)FNHA -15%wt TiO2.

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phenomenon of being doped, the magnified patterns of thementioned samples are also given in Fig. 5. It can be inferred fromthis figure that in addition to widening, the FNHA characteristicpeaks (reflections) gradually shifted to the right hand side. Thisstems from the reduction of the a-axis length of the hexagonal HAcrystals lattice due to lower ionic radii of F� (0.065 nm) comparedto OH� (1.32 nm) which introduces distortion into the lattice withincorporation of fluorine ions instead of hydroxyl groups in theapatite structure. This observation is in good agreement with thefact that fluorine addition tends to decrease the lattice parameter a,but does not obviously affect the lattice parameter c.

Also, FHA has the release of fluoride at a controlled rate whichcan lead to the provision of a strong bone with good mechanicaland functional properties [26]. Accordingly, different percentagesof TiO2 (5, 10, and 15) was added to FNHA and the producedcomposite was sintered by the TSS process. The FNHA-TiO2 nano-biocomposite samples after the TSS process are shown in Fig. 6. Ascan be seen in Fig. 6a, b and c, with increasing the TiO2 contentreinforced in the FNHA matrix, the compaction increases. Beforeexplaining the observed event, it is necessary to note that for pureFNHA the interconnection of the apatite particles during the TSSprocess yields high density of the sintered specimen. However, itwas reported that [27] with excess temperature, the decrease indensification is observed for pure FNHA. The reduction of the FNHAdensity can be attributed to the decomposition that results in the

formation of calcium phosphates as mentioned earlier. The aboveinterpretation will vary with the addition of TiO2 to FNHA. In fact,TiO2 can prevent early decomposition of FNHA and consequentlystrengthen the interactions between the apatite particles, whichleads to the increase of compaction as shown in Fig. 6. X-raydiffraction pattern of the sintered FNHA-TiO2 nanobiocomposite isillustrated in Fig. 7. The peaks relevant to FNHA and TiO2 as majorphases can be seen. One can also observe the TCP, CaF2 and CaTiO3phases. It is notable that when FNHA completely decomposes tocalcium phosphate phases, the density of composite begins todecrease. The formation of the CaTiO3 phases indicates that thecalcium phosphate phases have not been able to completely form.

TEM photographs of the sintered samples at various content ofTiO2 shown in Fig. 8a and b. As can be seen, there is almost nochange in the size of the grains by increasing the TiO2 content. Fromthe previous interpretations (explanations about Figs. 6 and 7) andalso TEM images, it can be concluded that TiO2 can only preventearly decomposition of FNHA but not change the FNHA grain size.This means that the compaction observed in Fig. 6 is not because ofthe decrease of the mean grain size but is because of the unde-composed FNHA resulted by the increase of TiO2. The formation ofthe CaTiO3 phases (see Fig. 7) indicates that TiO2 can successfullyprevent decomposition of FNHA to calcium phosphate phases.

The bioactivity of ceramics has been defined as “the bond abilitywith host bone tissue” [28]. The in vitro bioactivity is assessed by

Fig. 11. EDX analysis of samples with 10% and 15% TiO2 (points A, B in Fig. 10c, d); a) FNHA- 10%TiO2- point A; b) FNHA-10%TiO2- point B; c) FNHA-15%TiO2- point A.

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examination of the growth of bone like apatite on the surface of thesamples after soaking in Kokubo's SBF solution. The dissolutioncurves can be very helpful as they indicate the changes in calciumions concentration and the pH values versus the immersion time inSBF (Fig. 9). It is understood from this figure that the pH value aswell as the Ca ion concentration increased during the first twoweeks of experiments (Fig. 9a, b). Here, it can be claimed that the Caions concentration in fluorine natural hydroxyapatite (FNHA) has ahigher amount than that in the non-natural one (see XRF analysisfor NHA in Table 1), leading to the instability of the FNHA-TiO2nanobiocomposite and hence the entry of the calcium ions into theSBF solution. The event leas to the pH increase in the first twoweeks of experiments.

As can be observed in Fig. 9a, the Ca ions concentration in pureFNHA is higher than that in the FNHA-TiO2 nanobiocompositesamples. This event may be originated from the more density aswell as chemical stability of the FNHA-TiO2 nanobiocomposite andhence less reacting with the SBF solution in the first two weeks. Inother words, the release of the Ca ions from pure FNHA is higherthan that from the FNHA-TiO2 nanobiocomposite because of itslower chemical stability (see Fig. 6).

The release of the calcium ions from the surface into the SBFsolution leads to the formation of many silanol (SieOH) groups onthe surface. The silanol groups are heterogeneous nucleation sitesfor the apatite layers. As can be seen from Fig. 9a, the Ca ionsconcentration begins to decrease for pure FNHA and tends to befixed in the FNHA-TiO2 nanobiocomposite samples after twoweeks. This phenomenon could be explained by the notion that theSBF solution eventually becomes saturated from the Ca ions (after 2weeks), so that the calcium ions tend to leave the solution. In otherwords, a balance between the Ca ions release and Ca ions absorp-tion is achieved. It should be noted that there are two places for theabsorption of calcium ions. The first place is the surface of theapatite nuclei and the second place is the surface on which theapatite has not been formed. Obviously, the Ca ions concentrationin the surface of apatite nuclei is less than that in other surfaces andthat is why the Ca ions (in the form of CaP ions) in the solution havea tendency to migrate to the apatite nuclei. This is a mechanism forthe growth of the apatite layer in the SBF solution. It is necessary tonote that the SieOH nucleation sites also play a key role in thereduction of Ca ions. In other word, as these nucleation sites in-crease the absorption of calcium ions into these sites increases aswell, so that the concentration of the Ca ions in the SBF solution andconsequently the pH value are reduced (Fig. 9a, b). It is clear fromFig. 9b that the optimum amount of pH (close to the pH of humanblood plasma) has been achieved after 21 days for the FNHA-TiO2nanobiocomposite samples. The above results are confirmed bySEM images and the EDX analysis.

The SEM images of the FNHA- TiO2 nanobiocomposite samplesafter immersion in the SBF solution for 21 days are shown in Fig. 10.As can be seen, the pure FNHA has a lower ability in apatite for-mation compared to other samples composed of FNHA-TiO2(Fig. 10a). Fig. 10b shows that with increasing the TiO2 content to5 wt%, the apatite precipitations (light porous areas) haveincreased. According to Fig. 10 c and d, the apatite formation on thesurface of the samples continues with the increase of the TiO2percentage in the FNHA-TiO2 nanobiocomposite, which is attrib-uted to more availability of nucleation sites as mentioned in theprevious section.

The EDX analysis of the samples with 10% of TiO2 (points A and Bin Fig.10c) and 15% of TiO2 (point A in Fig.10d) is shown in Fig.11. Ascan be seen in this figure, the surface on which there is no theapatite layer (point B in Fig. 10c) has a higher Ca ions concentrationthan the surface on which the apatite layer is precipitated (point Ain Fig. 10c). This confirms what was stated in this study regarding

the growth of the apatite layer. On the other hand, the samples withhigher amount of TiO2 (point A in Fig. 10d) have more Si than thesamples with a lower amount of TiO2(see the EDX analysis of pointB and point A in Fig.10 c and d, respectively). This also confirms thatwith increasing the percentage of TiO2 up to 15, the SieOH nucle-ation sites also increase which leads to the ease of formation of theapatite layers and hence more bioactivity.

4. Conclusions

In the present study, an FNHA-TiO2 nanobiocompositeceramic with excellent biocompatibility and good chemical sta-bility was synthesized. The FNHA is completely formed at 8 h ofmilling, which implies that the doping process has beencompleted at or before this milling time. The SEM images of thesintered samples showed that with increasing the TiO2 contentreinforced in the FNHA matrix, the compaction increases,although the grain size remains constant. TiO2 can prevent earlydecomposition of FNHA by the formation of the CaTiO3 phase andhence strengthen the interactions between the apatite particleswhich results in the increase of compaction. The pH values aswell as the Ca ions concentration in the SBF solution haveincreased during the first two weeks of experiments due to moreCa concentration in natural-fluorhydroxyapatite than that in thenon-natural one, leading to the instability of the FNHA-TiO2nanobiocomposite and hence the entry of the calcium ions intothe SBF solution. The more Ca ions concentration in the SBF so-lution for pure FNHA compared to FNHA- TiO2 nanobiocompositesamples is related to the more density as well as chemical sta-bility of the FNHA- TiO2 nanobiocomposite and hence lessreacting with the SBF solution in the first two weeks of experi-ments. As the Ca ions concentration in the surface of apatitenuclei is less than that in other surfaces, the Ca ions in the SBFsolution migrate to the apatite nuclei. This is a mechanism for thegrowth of the apatite layer in the SBF solution. As the percentageof TiO2 increases, the SieOH nucleation sites also increase whichleads to the ease of formation of the apatite layers and hencemore bioactivity.

Acknowledgment

The authors would like to thank Dr. David Ogbemudia for hishelp in editing some of the figures.

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Introducing the fluorine doped natural hydroxyapatite-titania nanobiocomposite ceramic1. Introduction2. Materials and methods2.1. Preparation of the powder samples2.2. Synthesis of FNHA2.3. The composite formation2.4. The characterization procedure2.5. In vitro bioactivity evaluation

3. Results and discussion4. ConclusionsAcknowledgmentReferences