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Ga

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Annals of Anatomy xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Annals of Anatomy

j ourna l h omepage: www.elsev ier .de /aanat

ffects of fly ash and boric acid on Y2O3-stabilized tetragonal ZrO2

ispersed with MgAl2O4: An experimental study on rat subcutaneousissue

ulfem Erguna,∗, Metin Gurub, Ferhan Egilmeza, Isil Cekic-Nagasa, Dervis Yilmazc

Department of Prosthodontics, Faculty of Dentistry, Gazi University, Ankara, TurkeyDepartment of Chemical Engineering, Faculty of Engineering, Gazi University, Ankara, TurkeyDepartment of Oral and Maxillofacial Surgery, Faculty of Dentistry, Ankara, Turkey

r t i c l e i n f o

rticle history:eceived 3 November 2013eceived in revised form 12 February 2014ccepted 13 February 2014vailable online xxx

eywords:oric acidly ash

s u m m a r y

The aim of this study was to evaluate the subcutaneous tissue reaction around zirconia-based materials.Forty-eight male Wistar Albino rats were used in this study. Disk-shaped (1 mm height and 5 mm diam-eter) samples composed of 67% spinel (MgAl2O4), 27% tetragonal zirconia polycrystal, 4% (m/m) fly ashand 2% (m/m) boric acid were inserted into dorsal muscles of rats. After 1, 4, 8 and 16 weeks, the ani-mals were sacrificed and zirconia materials were removed with the surrounding tissue. Tissue sectionswere made with a microtome and then stained with hematoxylin and eosin. Sections were evaluated forthe intensity of inflammation. Additionally, the somatic and visceral lymph nodes were evaluated. Datawere submitted to one-way analysis of variance (ANOVA) and Tukey HSD tests at a significant level of

nflammatory reactionissue responseirconia

p < 0.05. There were statistically significant differences between mean inflammatory scores in differentexperimental periods (p < 0.05). In addition, the inflammatory reaction decreased over time. The testedmaterials had no damaging effect on the rat lymph nodes and did not have a toxic action on the inter-nal organs. Therefore, zirconia polycrystal tested in the present study may offer a promising treatmentalternative after further mechanical and biological studies are performed.

© 2014 Elsevier GmbH. All rights reserved.

. Introduction

Zirconia ceramics in dentistry has gained increased popular-ty due to its high biocompatibility, low bacterial surface adhesions well as improved mechanical and esthetic properties (Egilmezt al., 2013; Zhang, 2012). One of the main problems in this struc-ure is the progressive spontaneous transformation of zirconia fromhe metastable tetragonal phase into monoclinic (Egilmez et al.,013; Vagkopoulou et al., 2009). Additionally, during this trans-ormation a large volume increase (3–5%) occurs in the zirconiand the stresses occurred when cooling from the firing tempera-ure can be sufficient to crack the zirconia. Therefore, stabilizingxides such as CaO, MgO, Y2O3 or Ce2O3 are used for alloying pure

Please cite this article in press as: Ergun, G., et al., Effects of fly ash aMgAl2O4: An experimental study on rat subcutaneous tissue. Ann. An

irconia to maintain the tetragonal structure at room tempera-ure. Moreover, additions of these metal oxides and appropriateeat treatment can overcome this volume increase (Hanson et al.,

∗ Corresponding author at: Yesil Baris sitesi, No. 157, Alacaatlı, C ayyolu, Ankara,urkey. Tel.: +90 3122034191.

E-mail addresses: ergungulfem@yahoo.com, gulfem@gazi.edu.tr (G. Ergun).

ttp://dx.doi.org/10.1016/j.aanat.2014.02.003940-9602/© 2014 Elsevier GmbH. All rights reserved.

2000). A previous study reported improved mechanical and refrac-tory properties of spinel/zirconia composite structure especiallyafter firing at 1600 ◦C. Besides, these features are related to highdensification and good texture (Khalil, 2012).

Fabrication of large and complex shaped components from zir-conia is a both difficult and expensive process (Hanson et al., 2000).Therefore, the use of low cost materials such as fly ash, and boricacid mixed with spinel (MgAl2O4), could result in an economicadvantage.

Fly ash is a residue from coal based electric power plantscaught by cyclones at chimneys and is a mixture of compoundssuch as amorphous alumina, silica, ferric oxide, calcium oxide andother light metal oxides. In addition, it is the finest coal com-bustion residue (0.2–90 �m) with a specific surface area, typicallybetween 250 and 600 m2/kg. It is used in the field of cement andother construction materials, agriculture, metal recovery, waterand atmospheric pollution control, catalytic role in oxidation, chlo-

nd boric acid on Y2O3-stabilized tetragonal ZrO2 dispersed withatomy (2014), http://dx.doi.org/10.1016/j.aanat.2014.02.003

rination, synthesis of 2-mercaptobenzothiazole derivatives undermicrowave irradiation and as solid acid catalyst for esterificationreactions, for the synthesis of aspirin and oil of wintergreen (Khatriet al., 2010; Querol et al., 1997).

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2 G. Ergun et al. / Annals of Anatomy xxx (2014) xxx–xxx

Table 1Chemical composition of fly ash powder (%).

CaO

16.0

taautooBfim

iammcmrtta(

tnu

2

2

tflEMcwbNt

2

wGIwrl

2

(i

o

SiO2 TiO2 Al2O3 Fe2O3

47.08 1.03 21.96 6.48

Borate chemicals and boric acid are used in industrial applica-ions. Additionally, metal borates are not only found in nature butlso synthesized in the laboratory and have different applicationreas due to their useful properties (Qasrawi et al., 2005). Borax,lexite and colemanite are principally used in the ceramics indus-ry for manufacturing borosilicate and similar glasses. The presencef boric oxide in these glasses facilities the dissolution of metallicxides and produces a decrease in the melting point of the frits.oric oxide also influences both the refractive index and the coef-cient of expansion. The control of impurities of boron in otheraterials is also important (Sanchez-Ramos et al., 1998).Biocompatibility of a dental material is an important topic

ncluding several stages from in vitro tests through cell culturesnd in vivo experiments using animal and human clinical trials. Ani-al tests evaluate the biological effects of dental materials whichight be exerted on dental tissues and the risks that the material

an cause to health (Zhang et al., 2013). Furthermore, the animalodel is a prevalent tool in examining tissue responses and allergic

eactions to the material. The inflammatory response is a charac-eristic phenomenon common to all fibrous connective tissue andhe release of chemotactic mediators and growth factors may elicitnd sustain these inflammatory responses at the operation siteKaranth et al., 2013).

Based on these considerations, the present aim was to evaluatehe tissue biocompatibility of 3% yttria stabilized tetragonal zirco-ia mixed dispersed with MgAl2O4, boric acid and fly ash for dentalse in a rat dorsal muscle implantation test.

. Materials and methods

.1. Preparation of zirconia-based material

In order to see how the fly ash and boric acid affected the subcu-aneous tissue reaction around zirconia-based materials; 4% (m/m)y ash and 2% (m/m) boric acid (Yatagan coal powerplant andti Mining works, Mugla, Turkey) were added to the mixture ofgAl2O4 and tetragonal zirconia stabilized with yttria. The chemi-

al composition of fly ash (Yatagan coal powerplant and Eti Miningorks) is given in Table 1. The uniformity of mixture was supplied

y means of Spex-8000 ball milling (Spex Industries, Inc., Edison,J). Then this uniform mixture was pressed with 10 tons and sin-

ered by conventional methods.

.2. Experimental animals

After approval of the research protocol, this prospective studyas performed with the approval of the ethics committee of theazi University Hospital, in Ankara, Turkey (Process #025/2010).

n all, 48 male Wistar Albino rats weighing between 200 and 250 gere used in the present study. They were kept in an acclimatized

oom in a plastic cage and received a balanced diet and water adibitum.

.3. Surgery and drug administration

Rats were randomly divided into 2 groups: zirconia material

Please cite this article in press as: Ergun, G., et al., Effects of fly ash aMgAl2O4: An experimental study on rat subcutaneous tissue. Ann. An

n = 24) and sham operated (n = 24) (control) groups, to be exam-ned after 1, 4, 8 and 16 weeks from the surgical procedure.

The animals were anesthetized by intramuscular injectionf ketamine chlorhydrate (0.1 mg/ml) associated with xylazine

MgO NaO K2O SO3

3 2.12 1.28 0.93 2.13

(0.05 mg/ml), followed by shaving of dorsal fur, disinfection, inci-sion and divulsion of the subcutaneous tissue to insert the testmaterial that had a disk-shape (1 mm height and 5 mm diame-ter). To implant the tested materials (67% spinel, 27% tetragonalzirconia polycrystal, 4% fly ash and 2% boric acid) into the rat sub-cutaneous tissues, one pocket was created by blunt dissection inthe cranial portion to implant the test material in the subcutaneoustissue. After implantation, the wounds were sutured with 4-0 silkinterrupted sutures.

After 1, 4, 8 and 16 weeks, 6 animals were sacrificed ateach experimental period by anesthetic overdose. The materialswere explanted together with skin and connective tissue aroundthem for histological analysis. Following separation of the zir-conia from soft tissue carefully, the soft tissue samples wereimmersed immediately in buffered 10% formalin solution for 48 h.Then the samples were embedded in paraffin. Five micrometer-thick paraffin-embedded tissue sections were prepared with amicrotome (Leica SM-2000, Leica Corp., Germany). In addition,the sections were stained with hematoxylin and eosin for histo-morphological analysis and with Unna mast cell staining methodfor mast cell analysis. Then the sections were viewed by lightmicroscopy to determine if the staining in serial sections was con-sistent and to ensure that no serious artifacts (folds, tears, etc.) werepresent. A blind test was conducted at the same time using identicalstaff, equipment, and chemicals.

Tissue sections were evaluated microscopically by a pathol-ogist (Nikon Eclypse E-600, Nikon Corp, Japan) at 40×, 100×,200×, 400× and 600× magnification for the occurrence of inflam-matory response [0, no reaction—for absence of inflammatorycells (lymphocytes, plasmocytes, polymorphonuclear leukocytes,macrophages, and giant cells); 1, mild reaction—for presenceof mild chronic inflammatory cells; 2, moderate reaction—forpresence of moderate chronic inflammatory cells; 3, severereaction—for presence of an large number of inflammatory cells].

Evaluation of mast cells was carried out in microscopic fieldsadjacent to the test materials under a light microscope (LeicaDM4000 motorized light microscope) equipped with image ana-lyzer software (Leica QWin Plus, version 3.5.1, Leica Microsystems,Switzerland) at 200× magnification.

Additionally, the somatic (axillary) and visceral (mesenteric)lymph nodes from all rats were taken and washed together withskin samples in phosphate-buffered saline (PBS 0.1 M; pH = 7.4).Samples were fixed with 10% buffered formalin, embedded in paraf-fin, transversally sectioned at 5 �m and stained with hematoxylinand eosin. Capsular integrity, presence of lymph node hyperplasiaor tumor were evaluated under light microscope.

2.4. Statistics

One-way analysis of variance (ANOVA) and Tukey HSD testswere used for statistical analysis of inflammatory response at asignificance level of p < 0.05. Additionally, statistical differencesin mast cell number were investigated by Kruskal Wallis andMann–Whitney U tests at a significance level of p < 0.05.

3. Results

nd boric acid on Y2O3-stabilized tetragonal ZrO2 dispersed withatomy (2014), http://dx.doi.org/10.1016/j.aanat.2014.02.003

Notron activation and gamma spectrometry analysis results offly ash were given in Table 1. According to these analyses, in 1 gof fly ash, there was 0.37 Bq uranium activity. In brief, its annual

ARTICLE ING ModelAANAT-50845; No. of Pages 7

G. Ergun et al. / Annals of Anat

F1d

dee

4ttfwecgcsdr

sgcvo

c

F1d

ig. 1. Mean scores and standard deviation of inflammatory response after 1, 4, 8 and6 weeks following surgical procedure. *Bars with different letters are statisticallyifferent at p < 0.05.

osage unit equals to ≈4 × 10−3 mSv/y. Additionally, 27 ppm Th isquivalent to 0.1 Bq thorium activity and its annual dosage unitquals ≈1 × 10−3 mSv/y.

In all the rats studied (sham and zirconia groups for weeks 1,, 8 and 16), the material was inserted between muscle and fatissues. The intensity of inflammatory response in the connectiveissue around the zirconia material was analyzed in this studyor all experimental periods. Statistically significant differencesere observed between mean inflammatory scores at different

xperimental periods (p < 0.05). However, there were no signifi-ant differences between inflammatory scores of zirconia and shamroups (p = 1.000). Fig. 1 presents the mean values of histologi-al scores in different groups. After 1 week, the zirconia group’specimens showed grade 2–3 inflammation, which consisted of aense infiltration of inflammatory cells. Besides, the inflammatoryeaction decreased non-significantly over time (p = 0.970).

According to the statistical analysis of mast cell number, noignificant differences were observed between zirconia and shamroups (p = 0.794). In addition to this, a significant decrease in mastell number occurred with time (p < 0.05). Fig. 2 presents the meanalues of mast cell number in different groups. Photomicrographs

Please cite this article in press as: Ergun, G., et al., Effects of fly ash aMgAl2O4: An experimental study on rat subcutaneous tissue. Ann. An

f different inflammatory reactions are presented in Figs. 3 and 4.At the 1st week, in the operative region of both sham and zir-

onia groups, specimens showed mixed type inflammatory cell

ig. 2. Mean scores and standard deviation of mast cell number after 1, 4, 8 and6 weeks following surgical procedure. *Bars with different letters are statisticallyifferent at p < 0.05.

PRESSomy xxx (2014) xxx–xxx 3

infiltration. In addition, this region marked proliferation of granula-tion tissue characterized by giant cells. The histological evaluationshowed that all the materials caused moderate and severe inflam-matory reaction around the materials at 7 days, which decreased inthe following weeks. Moreover, there were no eosinophilic leuko-cytes. Two specimens of Group Sham and 3 of group zirconia hadfibrin exudates surrounding the material. Regarding the number ofmast cells, Group zirconia had fewer mast cells than that of GroupSham.

After 4 weeks, both sham and zirconia groups had a decreasein concentration of inflammatory reaction around the materialswhen compared with 1st week. While neutrophile leucocytes werenot detected, lymphocytes were the major components of theinflammatory cell reaction. The operative sites were covered byan epithelium. In some of the specimens, fibrin exudate aroundthe material had completely disappeared. Additionally, in all of thesamples, an acellular fibrous band was observed surrounding thematerials. Mast cells show a preference toward perivascular local-ization within tissue near the material and the numbers of the cellswere lower than that of week 1.

All of the specimens evaluated after 8 and 16 weeks dis-played similar histological appearance. Moreover, inflammatorycell reaction decreased and was observed around the hair follicles.Fibrous bands surrounding the materials were thickened, whencompared with week 4 specimens. However, the number of mastcells decreased.

3.1. Lymph node evaluation

In all of the groups, the somatic and visceral lymph nodes oftreated animals displayed a normal structure of the parenchymawith prominent germinal centers. The capsular integrity was pre-served in all of the specimens and no lymph node hyperplasia ortumors were observed under light microscope. Additionally, nomacrophages or plasma cells were found (Fig. 5). In zirconia andsham groups, the lymph node of treated rats displayed a normalstructure, but with numerous mast cells.

After 1 week; mixed type inflammation (acute inflammation)was detected both in sham and zirconia groups. In addition, in week4; in both of the groups, while the acute inflammatory cell numberdecreased, chronic inflammatory cells (lymphocytes) increased incomparison to the 1st week.

After 8 weeks, loose connective tissue was observed around thematerial. Additionally, in the sham group; inflammation decreased.In week 16, collagen content increased around the material whencompared to the 8th week. However, fibrous band like connectivetissue was observed in the sham group samples.

4. Discussion

The biocompatibility characteristics of the materials used forprosthetic restorations should be investigated, since the toxic com-ponents present in these materials could produce irritation or evendegeneration of the surrounding tissue (Ballo et al., 2009; Ergunet al., 2011; Garvie et al., 1984; Ikarashi et al., 2005). Additionally,the biocompatibility of a material is important, especially if it isto be used in implant devices (Garvie et al., 1984; Ikarashi et al.,2005). The subcutaneous implantation technique performed in thepresent study was firstly introduced by Torneck (1966). Besides, itis one of the most suitable methods to determine the local effectsand biocompatibility of dental materials on the skin and lymphatic

nd boric acid on Y2O3-stabilized tetragonal ZrO2 dispersed withatomy (2014), http://dx.doi.org/10.1016/j.aanat.2014.02.003

organs of rats prior to clinical studies (Rezzani et al., 2004). Previousstudies evaluated tissue response by implanting zirconia ceramicsto the dorsal muscle of the rats (Garvie et al., 1984; Ikarashi et al.,2005). Therefore, in the present study, the biocompatibility of %3

Please cite this article in press as: Ergun, G., et al., Effects of fly ash and boric acid on Y2O3-stabilized tetragonal ZrO2 dispersed withMgAl2O4: An experimental study on rat subcutaneous tissue. Ann. Anatomy (2014), http://dx.doi.org/10.1016/j.aanat.2014.02.003

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Fig. 3. Photomicrographs of skin and connective tissue of sham operated rats. (a, c, e and g) Hematoxylin and eosin staining showing inflammatory response, at weeks 1, 4,8 and 16, 200× magnification, (b, d, f and h) mast cells, 400× magnification. Arrows indicate mast cells.

Fig. 4. Photomicrographs of skin and connective tissue of zirconia placed rats. (a, c, e and g) Hematoxylin and eosin staining showing inflammatory response at weeks 1, 4,8 and 16, 200× magnification, (b, d, f and h) mast cells, 400× magnification. Arrows indicate mast cells.

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G. Ergun et al. / Annals of Anatomy xxx (2014) xxx–xxx 5

F d shan

ybm

iSs1iiMedTpw

ptHaptrpshgsbg

isp(gfpt

l

ig. 5. Photomicrographs of somatic and visceral lymph nodes of zirconia placed anode evaluation, 200× magnification.

ttria stabilized tetragonal zirconia mixed dispersed with MgAl2O4,oric acid and fly ash for dental use were evaluated in rat dorsaluscle.Additionally, male rats were used in the previous animal

mplantation studies (Rezzani et al., 2004; Kurita et al., 2013).ince males are generally twice the size of female rats, their cross-ectional and cortical areas are larger than females (Turner et al.,990). Furthermore, a previous study by Ashcroft et al. (2003)

ndicated that estrogen replacement might have effects on ovar-an hormone deficiency on wound contraction in a female rat.

oreover, a previous study by Tateishi et al. (2013) reported that,strogen deficiency might have affected bone healing and boneensity around titanium implants, especially in cancellous bone.hus, for using the dorsal muscle of rat efficiently and to avoidotential hormonal effects of female rats on implantation, male ratsere used in the present study.

A previous study compared the tissue response of Ti–Zr alloy,ure Ti, Zr by an implantation test in animal bodies and reported nooxicological change in body or organ weights (Ikarashi et al., 2005).owever, an inflammatory response occurred by the increase inctivities of fibroblasts, lymphocytes, and other cells. Similarly, arevious study by Rezzani et al. (2004) evaluated the implanta-ion of different alloys and indicated the clear rapid inflammatoryesponse in the skin and lymph nodes. In accordance with therevious studies, after 1 week from implantation, zirconia group’specimens received a dense infiltration of inflammatory cells. Theistological evaluation showed that the implantation lead to arade 2–3 inflammatory reaction at 7 days and showed a non-ignificant decrease with time (Fig. 1). Additionally, the differencesetween the mean inflammatory scores of zirconia and the shamroups were significant in different experimental periods.

Mast cells found in connective tissue play an important rolen acute inflammation owing to their release of stored and newlyynthesized inflammatory mediators (Coussens et al., 1999). In theresent mast cell analysis, mast cell number decreased with timeFig. 2). However, there were no significant differences between theroups. Similarly, a previous study investigated the effects of alloysor ceramic crowns and removable prostheses on the skin and lym-

Please cite this article in press as: Ergun, G., et al., Effects of fly ash aMgAl2O4: An experimental study on rat subcutaneous tissue. Ann. An

hoid organs of rats and indicated that mast cells decreased overime (Rezzani et al., 2004).

Previous studies reported that the accumulation of eosinophiliceukocytes is a feature of inflammatory reactions that occur in aller-

m operated rats. Hematoxylin and eosin staining at weeks 1, 4, 8 and 16 for lymph

gic reactions. Additionally, they act as a modulator in the allergicinflammatory reactions (Gonzalo et al., 1996; Hubscher, 1975). Inthe present study, no eosinophilic leukocytes were detected in anyof the specimens. According to the histological analysis, the surgi-cal procedures and implantation resulted in an inflammation ratherthan creating an allergic reaction.

The zirconia material in the subcutaneous area did not changeposition and cause sensitivity or mobility reactions. Locally, whentouching the area, the material was palpated as a non-painful nod-ule. In addition, in all of the samples, an acellular fibrous band,similar to a capsule was observed which surrounded the materialsand isolated the product. The examination of the area showed thatthe zirconia did not have a toxic or irritating components causingallergic reaction on the tissue, which could lead to rejection or elim-ination of the material. A previous study by Christel et al. (1989)investigated mechanical properties and short-term in vivo evalua-tion of yttrium-oxide-partially stabilized zirconia and reported thatzirconia polycrystals (Y-PSZ) tended to become encapsulated byfibrous tissue as observed for alumina control samples suggestingthat zirconia was biocompatible.

Previous studies indicated that Zr compounds have no local orsystemic toxic and negative effects on soft and hard tissues (Piconiand Maccauro, 1999). Furthermore, zirconia usually has smoothsurfaces and manages to regulate immunity, vesicular transportand cell cycle regulation (Mai et al., 2012).

Recently, manufacturers are looking for cheaper, more efficientways of producing dense stabilized zirconia ceramics (Butler andDrennan, 1982). Therefore, the addition of low cost materials (inthe current study, such as MgAl2O4, boric acid and fly ash) tothe base structure could be an alternative for fabrication of eco-nomic restorations. Furthermore, for controlling the stress-inducedtetragonal-to-monoclinic phase transformation, crack propagationshould be arrested by achieving high toughness. The additives(MgO, CaO, CeO) decrease the transformation temperature, allow-ing the zirconia to be cooled to room temperature retaining a (meta)stable cubic phase over the entire temperature range (Hanson et al.,2000). In the present study, MgAl2O4 was used as the base mate-rial for reducing the cost of the zirconia. Several researchers dealt

nd boric acid on Y2O3-stabilized tetragonal ZrO2 dispersed withatomy (2014), http://dx.doi.org/10.1016/j.aanat.2014.02.003

with the spinel/zirconia composites due to their superior ther-momechanical properties. Moreover, spinel composite systemsare effective in strengthening and toughening the ceramic matrix(Khalil, 2012). Besides, a previous study by Morita et al. (2002)

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6 G. Ergun et al. / Annals of Anat

Table 2Notron activation and gamma spectrometry analysis results of fly ash.

Element Gamma spectrometryanalysis

Notron activationanalysis

rmo

shtspamce(athibio(paft2gw

lierrcbttcitp

anpctcotetolaF

reaction and sensitization to Ti–Zr alloy compared with pure Ti and Zr in rat

Uranium 32 ppm 26 ppmThorium 27 ppm 27 ppm

eported enhanced superplasticity of MgAl2O4 in Y-TZP. Furtherechanical tests should be performed to test enhanced properties

f this new material.In a previous study by Khatri et al. (2010), the use of fly ash

upport not only reduced the cost of the catalyst, it also showedigh acidity and catalytic activity for benzylation of benzene andoluene. Furthermore, they concluded that fly ash can replace pureilica after its suitable activation and could be used as a solid sup-ort for loading of zirconia. Fly ash used in the present study is

coal burning by-product that might increase the level of toxicetals and ionizing radiation in the living environment by con-

entrating elements like uranium, thorium, and potassium (Fucict al., 2011). In contrast, a previous study by Zielinski and Finkelman1997) reported that, limited measurements of dissolved uraniumnd radium in water leachates of fly ash and dissolved concen-rations of these radioactive elements are below levels of humanealth concern. Similarly, a previous study by Fucic et al. (2011)

ndicated that radioactive elements in coal and fly ash should note sources of alarm. Besides, the vast majority of coal and the major-

ty of fly ash are not significantly enriched in radioactive elements,r in associated radioactivity, compared to common soils or rocksZielinski and Finkelman, 1997). Based on these arguments, in theresent study, only a small amount of fly ash (4%) has been used. Theverage value of estimated external effective dose rate is 1.0 mSv/yor members of the general public recommended by the Interna-ional Commission on Radiological Protection (ICRP) (Tahir et al.,006). Therefore, according to the notrone activation analysis andamma spectrometry, the radioactivity content of the fly ash usedere below levels of human health concern (Table 2).

Although boric acid was widely used therapeutically in the past,ater, it resulted in significant toxicity. Then, in the past few yearsts usage was limited. A previous study by Heindel et al. (1992),valuating toxicity of boric acid in mice and rats, indicated occur-ence of developmental toxicity below maternally toxic levels inats. In accordance with the previous study, Weir and Fisheri (1972)oncluded that both compounds (boric acid and borax) at 350 ppmoron equivalent had no adverse effect on fertility, lactation, lit-er size, weight and appearance of rats. Boric oxide used at 2% inhe current study acts as a glass former and is used as an acidiconstituent of glazes. It is the mineral colemanite and practicallynsoluble and raw when none-fritted. It has an important func-ion as a flux in addition to reduce viscosity of molten glazes andromoting high gloss (Weir and Fisheri, 1972).

Even the conventionally approved zirconia materials could have completely different composition; there is no standard zirco-ia material which can be used as control material. Therefore, theresent study has the limitation of not using the un-modified zir-onia as control material. Additionally, in implantation studies, theraumatic injury due to experimental procedures and the surgi-al process of implantation might be responsible for the initiallybserved inflammatory reaction (Gomes-Filho et al., 2008). In addi-ion to the zirconia insertions, a sham operation was performed onach rat in the control group. Another limitation concerns the facthat the limited number of animals used in animal studies because

Please cite this article in press as: Ergun, G., et al., Effects of fly ash aMgAl2O4: An experimental study on rat subcutaneous tissue. Ann. An

f ethical and economical reasons. Since the number of animals wasimited in the present study, we have used previous zirconia studiess a reference. This is a preliminary study for the tested material.or future in vivo and mechanical studies, this is an exciting new

PRESSomy xxx (2014) xxx–xxx

field of research aimed at demonstrating the properties of our newzirconia material containing different percentages of boric acid andfly ash with improved structure.

Within the limitations of this in vivo study, the following con-clusions could be drawn:

1. The tested materials had no damaging effect on the rat lymphnodes and did not have a toxic or allergic reaction in the internalorgans.

2. With the decrease in inflammation, mast cell number is alsodecreased. Therefore, this procedure resulted in an inflammationrather than creating an allergic reaction.

3. Zirconia polycrystal tested in the present study may offer apromising treatment alternative. However, further mechanicaland biological studies are needed to demonstrate their feasibilityand efficacy.

Acknowledgement

This study was financially supported by the Scientific ResearchProjects Fund of Gazi University.

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