Bioactivity and mineralization of hydroxyapatite with bioglass as sintering aid and bioceramics with Na 3Ca 6(PO 4) 5 and Ca 5(PO 4) 2SiO 4 in a silicate matrix

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Bioactivity and mineralization of hydroxyapatite with bioglass as sintering aid andbioceramics with Na3Ca6(PO4)5 and Ca5(PO4)2SiO4 in a silicate matrix

Hande Demirkiran a, Arunesh Mohandas b, Motokazi Dohi b, Alonso Fuentes b,Kytai Nguyen b, Pranesh Aswath a,⁎a Materials Science and Engineering Department, University of Texas at Arlington, Arlington, TX 76019, U.S.A.b Bioengineering Department, University of Texas at Arlington, Arlington, TX 76019, U.S.A.

a b s t r a c ta r t i c l e i n f o

Article history:Received 6 May 2009Received in revised form 25 September 2009Accepted 27 October 2009Available online 1 November 2009

Keywords:HydroxyapatiteSilicatesBioactivityMineralization

Hydroxyapatite and Bioglass®-45S5 were sintered together creating new ceramic compositions that yieldedincreased apatite deposition and osteoblast differentiation and proliferation in vitro compared tohydroxyapatite. The sintered products characterized by X-ray diffraction, revealed hydroxyapatite as themain phase when small quantities (1, 2.5 and 5 wt.%) of bioglass was added. Bioglass behaved as a sinteringaid with β-TCP (Ca3(PO4)2) being the minor phase. The amount of β-TCP increased with the amount ofbioglass added. In compositions with larger additions of bioglass (10 and 25 wt.%), new phases withcompositions of calcium phosphate silicate (Ca5(PO4)2SiO4) and sodium calcium phosphate (Na3Ca6(PO4)5)were formed respectively within amorphous silicate matrices. In vitro cell culture studies of the ceramiccompositions were examined using bone marrow stromal cell (BMSC). Cell proliferation and differentiationof bone marrow stromal cells into osteoblasts were determined by Pico Green DNA assays and alkalinephosphatase (ALP) activity, respectively. All hydroxyapatite–bioglass co-sintered ceramics exhibitedlarger cell proliferation compared to pure hydroxyapatite samples. After 6 days in cell culture, the ceramicwith Ca5(PO4)3SiO4 in a silicate matrix formed by reacting hydroxyapatite with 10 wt.% bioglass exhibitedthe maximum proliferation of the BMSC's. The ALP activity was found to be largest in the ceramic withNa3Ca6(PO4)5 embedded in a silicate matrix synthesized by reacting hydroxyapatite with 25 wt.% bioglass.

© 2009 Elsevier B.V. All rights reserved.

1. Introduction

Hydroxyapatite (Ca10(PO4)6(OH)2) is one of the most well knownphosphates in the biologically active phosphate ceramic family byvirtue of its similarity to natural bone mineral. Synthetic andbiologically harvested hydroxyapatite finds a variety of biologicalapplications and elicits the formation of an apatite layer at theinterface with bone tissue [1–5]. Applications include bone repair inprosthetics [6], and dental applications [7].

Although hydroxyapatite has an osteoconductive nature andsupports osteogenic differentiation of marrow stromal cells (MSCs)[8], its bioactivity, while attractive, is not nearly sufficient to activelyform apatite layers on surfaces [9–11]. In addition, hydroxyapatiterequires longer time and large number of MSCs to differentiate theminto osteoblasts [8].

Other materials such as phosphate ceramics and different bioglasscompositions have been used as sintering aids to assist in sintering ofhydroxyapatite or as a component of composite to enhance materialproperties including bioactivity. For instance, Chatzistavrou et al.

found that sintered hydroxyapatite composites containing differentamounts of Bioglass®45S5 lead to increased bioactivity [12]. Thesodium phosphates have also been found to be effective sintering aidsfor hydroxyapatite, and resulting products were found to be bioactive[13]. In toxicity studies of silica containing calcium phosphate glasses,it has been shown that the presence of even 5 wt.% SiO2 in a glassresults in significant mortality when injected intraperitoneally intomice [14]. In another study, a calcium phosphate phasewas formed onthe surface of calcium phosphate invert glasses of composition60CaO.30P2O57Na2O3TiO2 when immersed in simulated body fluid(SBF) [15]. Tricalcium phosphate (TCP) a crystalline bioactive ceramicalso degrades to calcium and phosphate salts both in vivo and in vitro,and also results in the precipitation of hydroxyapatite on the surfaceof an implant [16]. Several studies with β-TCP and biphasic HA/β-TCPceramics have shown that β-TCP behaves in an osteoinductivemanner enhancing bone in-growth around and into implants inanimal studies [17,18]. In a separate study using a mixture ofhydroxyapatite with TCP with autogenous cancellous bone it wasshown that there was strong callus formation and a strong bony unionafter four weeks of implantation [19]. Among all the calciumphosphates used in biological applications only two calcium phos-phates are stable when they are in contact with aqueous media: At apHb4.2 the stable phase is CaHPO4∙2H2O (DCP) while at pHN4.3 the

Materials Science and Engineering C 30 (2010) 263–272

⁎ Corresponding author. Materials Science and Engineering Department, 500 WestFirst Street, Rm. 325, Arlington, TX 76019, United States. Tel.: +1 817 272 7108.

E-mail address: [email protected] (P. Aswath).

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stable phase is hydroxyapatite [20]. Furthermore, different calciumphosphates have different induction time for the formation ofcarbonated apatite with some of the calcium deficient hydroxyapatiteexhibiting poor bioactivity while the TCP and calcium pyro phos-phates exhibiting very good bioactivity [20].

Amongall bioglass compositionsBioglass®45S5developedby Larry L.Hench [21] consisting of 45 wt.% SiO2, 24.5 wt.% Na2O, 24.5 wt.% CaO,and 6 wt.% P2O5 has been found to be one of the most bioactive glasses[11]. Moreover, it has the potential to be used in many more bioactiveapplications than hydroxyapatite [22]. The bioactivity of Bioglass®45S5was shown to be insensitive to the level of crystallization with the 100%crystalline material exhibiting the same level of bioactivity as theamorphous material [23]. In a bioactivity study an increased cytotoxicresponse was shown for glasses with very high sodium oxide (Na2O)content. This increase in cytotoxic response was attributed to theincrease in local pHdue to ion exchange reaction occurring at the surfaceof the bioactive glass [24].

The Bioglass®45S5 by itself is not structurally viable and as astructural material sintered hydroxyapatite is a very brittle ceramicwhich is more or less strong in compression and week in tension [25].Therefore, Bioglass®45S5 is used in conjunction with hydroxyapatiteeither as a sintering aid (b5 wt.%) or second phase (N5 wt.%) [2,12,26]which may also result in improved mechanical properties specificallycompressive strength. There have been many attempts to combinehydroxyapatite with bioactive glasses of different compositions inorder to produce composites with improved mechanical andbiological properties [11,12,27,28] and significant gains in mechanicalproperties with improved bioactivity have been reported.

In the present study, a series of hydroxyapatite–Bioglass®45S5mixtures was sintered yielding products ranging from sinteredhydroxyapatite with β-TCP to new chemistries of ceramics withcompositions of calcium phosphate silicate to sodium calciumphosphate in silicate matrices. The uniqueness of this study is in thedevelopment of crystalline calcium phosphate silicate and sodiumcalcium phosphate phases within an amorphous silicate matrix. Thesecrystalline phases embedded within a silicate matrix have not beenevaluated previously for their bioactivity. The only prior studiesinvolving these chemistries (high Bioglass content) are a sinteringstudy of hydroxyapatite with Bioglass® [27], and a bone cement thatcontained sodium calcium phosphate [29] and a sintering study of β-TCP with Na4P2O7∙10H2O that yielded sodium calcium phosphate asone of the phases [30].

The structure and composition of the new chemistry wereexamined using X-ray diffraction and scanning electron microscopy.Mechanical properties were evaluated using compression tests. Inaddition, rat bone marrow stromal cells were cultured on thesesubstrates, and the cell proliferation and osteoblast differentiationafter 3 and 6 day cell culture were investigated using Pico Green DNAassays and alkaline phosphatase activity, respectively.

2. Experimental procedure

2.1. Formulation of hydroxyapatite–Bioglass®45S5 blends

2.1.1. Powder preparationThe bioglass powder consisting of 45 wt.% SiO2, 6 wt.% P2O5,

24.5 wt.% Na2O, and 24.5 wt.% CaO also known as Bioglass®45S5 wasacquired from US Biomaterials with a particle size of b90 µm, and thehydroxyapatite with a chemical composition of Ca10(PO4)6OH2 wasacquired from Alfa Aesar with a particle size of b44 µm. Five differentmixtures were prepared with 1, 2.5, 5, 10, and 25 wt.% Bioglass®45S5addition to hydroxyapatite.

2.1.2. Sample preparationThe Bioglass®45S5 powders were mixed with a proper amount of

hydroxyapatite in 250 ml polyethylene bottles, and ball milled for

30 h with acetone. After ball milling, the mixtures were dried in theoven at 80 °C for 24 h. The dried powder mixtures were sieved untilthe particles were separated from each other. The powders werepressed uniaxially in a die with a diameter of 12.7 mm to a pressure105 MPa, and sintered at 1200 °C for 4 h with a heating rate of 4 °C/min. The sintering was followed by cooling at 10 °C/min down toroom temperature.

2.2. Material characterization

2.2.1. Density and porosity measurementsThe apparent densities of the green and sintered bioceramic

samples were determined by a simple geometric weight over volumemethod. 8 to 10 samples were used to determine the average densityfor each group. The theoretical densities of each groupwere calculatedby rule of mixtures. The porosity of the sintered product wasmeasured by mercury porosimetry using a Quantachrome Instru-ments Poremaster Mercury Porosimeter.

2.2.2. X-ray diffraction (XRD) analysesX-ray diffraction studies of sintered Bioglass®45S5, hydroxyapa-

tite, and hydroxyapatite–Bioglass®45S5 mixtures were examinedusing a Siemens Kristalloflex 810 Powder Diffractometer using CuKα

radiation. The data were recorded over the 2θ range of 20–60° with a0.01° step size and a count time of 0.1 s.

2.2.3. Scanning electron microscopy (SEM)The development of microstructures in the bioceramic samples

after sintering and subsequent cell culture studies was investigatedusing Hitachi S-300N and Zeiss Supra 55VP scanning electronmicroscopes operated in secondary electron mode.

2.2.4. Compression testThe compression test was performed with MTS Q-Test150 with a

150 kN capacity. Five different (1, 2.5, 5, 10, and 25 wt.%) hydroxy-apatite–Bioglass®45S5 cylindrical bioceramic samples were preparedaccording to ASTM C773-88 (2006) Standards with approximately 2.0mean diameter/height ratio. The tests were run at a crosshead speedof 0.5 mm/min. Compressive strength of cylindrical samples wasmeasured reporting load to failure divided by the cross-sectional areaof the samples. The number of specimens tested for each group was atleast 3.

2.3. Bioactivity (in vitro) characterization

2.3.1. Isolation and culture of bone marrow stromal cellsPrimary rat bone marrow stromal cells (BMSCs) were isolated

from the femurs of Sprague–Dawley rats. The rats were sacrificedwithCO2 and the femurs were excised. Aseptically, the epiphyses wereremoved and the diaphyses were flushed with 5 ml of complete bonemedia (DMEM) from Invitrogen, supplemented with 10% fetal bovineserum (FBS) from Hyclone, 0.28µM L-ascorbic acid from RPI Corp.,10 mM β-glycerol phosphate from Sigma, 10 nM dexamethasonefrom Sigma, 1% penicillin–streptomycin and 0.1% fungizone fromInvitrogen using an 18 gauge needle. Aspirating the pellets up anddown using the syringe broke up the bone marrow pellets, and thecells were seeded in T-25 flasks (~1 femur per T-25 flask). The T-25flasks were incubated overnight at 37 °C in 5% CO2. After 24 h non-adherent cells were removed by 4–5 washes with phosphate buffersolution (PBS) and the adherent cells were further cultured incomplete bone media. Upon confluence, cells were detached fromthe culture surface with trypsin-EDTA, centrifuged at 1000g for10 min, and re-suspended in complete bone media for use inexperiments or subculture. For analysis, cells on the ceramic diskswere lysed on day 3 and day 6 with 1 ml of 1% Triton X100. Theexperiments were performed on triplicate samples for each group,

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and cells were seeded on the samples at a density of 1×104 cells/cm2

and incubated at 37 °C.

2.3.2. Cell proliferationA Pico Green DNA assay kit (Molecular Probes, Eugene, OR) was

used to quantitatively determine the amount of total DNA that iscorrelated to the number of cells grown on the hydroxyapatite–Bioglass®45S5 substrates. Following the manufacturer's instruction,100 µl aliquots of each cell lysate sample was added to 400 µl of1×TE in a cuvette. PicoGreen Reagent Dye solution (500 µl) wasadded to each cuvette and incubated at room temperature for 2–5 min. A blank sample and standards containing final concentrationsof 0 to 25 ng/ml of calf thymus DNA were used to make a DNAstandard curve. After the incubation time, readings of samples andstandards were taken with a fluorometer (VersaFluor Fluorometer,BioRad) with emission wavelength at 480 nm and excitationwavelength of 520 nm.

2.3.3. Alkaline phosphatase activityAlkaline phosphatase is an early marker of osteoblast differenti-

ation that was measured by a previously reported method [31]. Thequantification of alkaline phosphatase is achieved by the conversionof p-nitrophenyl phosphate, a colorless substrate, into p-nitrophenol,a yellow-colored substance. 80 µl aliquots from each lysate samplewere added to individual wells in a 96-well plate with 100 µl ofsubstrate solution (4 mg/ml) and 20 µl of alkaline buffer (1.5 M 2-amino2methyl-1-propanol at a pH of 10.3). The plate was thenincubated for 1 h at 37 °C, and the reaction was stopped by adding100 µl of 0.3 N NaOH. The plate reader (UV max kinetic microplatereader, Molecular Devices) was used to determine the absorbance at405 nm. Samples were compared to p-nitrophenol standards rangingfrom 0 to 500 µM.

2.3.4. Inductive coupled plasma (ICP) analysisICP analysis was performed by Chemical Solutions Ltd. Calcium,

phosphorous, potassium, and magnesium concentrations in thecomplete bone media that was taken from the original media and6 day cell culture of hydroxyapatite and 1, 2.5, 5, 10, and 25 wt.%Bioglass®45S5 added hydroxyapatite bioceramics were measured inorder to determine the ion leaching from the substrate to thesolutions.

2.4. Statistics

The in vitro bone marrow stromal cell proliferation and differen-tiation tests were performed on triplicate samples for each group ofbioceramic compositions. The data represented as mean±SD (Stan-dard Deviation). Statistical difference was analyzed using one-wayANOVA variance analysis, and p value of less than 0.05 was consideredsignificant.

3. Results

3.1. Physical and chemical characterization

Five different compositions ranging from hydroxyapatite with1 wt.% Bioglass®45S5 to a composition with 75 wt.% hydroxyapatiteand 25 wt.% Bioglass®45S5 were sintered at 1200 °C for a period of4 h. Shown in Fig. 1 is the XRD patterns of the five different ceramicsand the analysis of the structure is listed in Table 1. The primaryceramic present when 1, 2.5 and 5 wt.% Bioglass® was sinteredwith hydroxyapatite is hydroxyapatite (JCPDS#09-0432) with β-TCP(Ca3(PO4)2, JCPDS#09-0169) as the secondary phase. There is no evi-dence of any crystalline silicate phase formed using X-ray diffraction.This indicates that the Bioglass®45S5 behaves more as a sintering aidand promotes the conversion of HA to β-TCP.

In order to estimate the extent of conversion of HA to β-TCPquantitative X-ray diffraction techniques were used. Referencesamples of known amounts of hydroxyapatite and β-TCP (up to 60%β-TCP) were prepared and homogenized and their powder X-raypatternswere determined. The dominant peaks in hydroxyapatite, the(2,1,1) peak and the (0,2,10) peak in β-TCP were used as reference forquantitative analysis. The ratio of integrated intensity of the (0,2,10)β-TCP peak to the (2,1,1) hydroxyapatite peak to the wt.% of β-TCP isshown in Fig. 2 and is mathematically represented in Eq. (1).

Iβ�TCPð0;2;10Þ.

IHAð2;1;1Þ= 0:0174Wβ�TCP−0:0976 ð1Þ

The ratio was used in each case to determine the ratio of crystallinephase β-TCP to hydroxyapatite in the sintered composites andsuperimposed on the calibration curve are the actual ratios for theceramics with up to 5 wt.% Bioglass®45S5. It is evident from Fig. 2 thatas the extent of Bioglass®45S5 is increased the extent of conversion ofhydroxyapatite to β-TCP increases till it reaches 35% at a compositionof 5 wt.% Bioglass®45S5.

However, when 10 and 25 wt.% Bioglass®45S5 are added to thehydroxyapatite and sintered, themain crystalline phase present in thesintered product is no longer hydroxyapatite. It is calcium phosphatesilicate (Ca5(PO4)2SiO4, JCPDS#40-0393) when 10 wt.% Bioglass®45S5is added, and sodium calcium phosphate (Na3Ca6(PO4)5, JCPDS#11-0236) when 25 wt.% Bioglass®45S5 is used.

Fig. 1. Powder X-ray diffraction spectra of bioceramics formed by sintering hydroxy-apatite with 1, 2.5, 5, 10, and 25 wt.% Bioglass® at 1200 °C for 4 h.∇: (Ca10(PO4)6(OH)2),↓: β-Ca3(PO4)2, □: Ca5(PO4)2SiO4, and 0: Na3Ca6(PO4)5.

Table 1Bioceramic compositions formed by sintering hydroxyapatite with 1, 2.5, 5, 10, and25 wt.% Bioglass®45S5 at 1200 °C for 4 h.

Bioglass® (wt.%) Composition of phases present

0 Synthetic hydroxyapatite (Ca10(PO4)6OH2)1 Synthetic hydroxyapatite+β-TCP (β-Ca3(PO4)2)2.5 Synthetic hydroxyapatite+β-TCP5 Synthetic hydroxyapatite+β-TCP10 Calcium phosphate silicate (Ca5(PO4)2SiO4)+β-TCP+

amorphous silicate phase25 Sodium calcium phosphate (Na3Ca6(PO4)5)+amorphous

silicate phase

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3.2. Porosity and density

The theoretical, green, and sintered densities of the bioceramiccompositions are shown in Fig. 3(a) and the porosity contentmeasured by mercury porosimetry of the sample cross-section isshown in Fig. 3(b). The theoretical density is based on a simple rule ofmixtures with the assumption that constituents used in the mixremain unchanged on sintering, which is certainly not the case here,but yet provides a first estimate of the density of the ceramic product.Bioglass®45S5 has lower density compared to synthetic hydroxyap-atite, and as expected the density of the mixtures increases aftersintering and decreases with an increase in Bioglass®45S5 content.However, the observed decrease in density is larger than thatpredicted by simple rule of mixtures. In compositions with up to5 wt.% Bioglass®45S5 up to 35% of the hydroxyapatite is decomposedto β-TCP, the theoretical density of hydroxyapatite is 3.16 g/cm3 [40]and that of β-TCP is [41] 3.07 g/cm3. The lower density of β-TCPcompared to hydroxyapatite partly accounts for the decrease indensity of compositions with up to 5% Bioglass®45S5 but does notcompletely explain it. The porosity content of the ceramics with up to5 wt.% Bioglass®45S5 as measured by mercury porosimetry is close to0% (Fig. 3(b)) which indicates that there is some closed porosity in thesintered product resulting in final densities ranging between 85 and90% of theoretically calculated values.

Compositions with higher amounts of Bioglass®45S5 of 10 and 25%exhibited higher levels of porosity (14 and 10% porosity after sinteringrespectively) and lower levels of densification indicating the presenceof glassy phases that retarded the densification process. This alsoexplains the fact that the measured density of the finished ceramic inthe compositions with 10 and 25 wt.% Bioglass® is significantly lowerthan the theoretical density.

3.3. Microstructural characterization

The secondary electron scanning electron micrographs (SEM) ofhydroxyapatite with 5, 10 and 25 wt.% Bioglass®45S5 after sinteringare shown in Fig. 4(a–c), respectively. Fig. 4(a) shows the micro-structure of hydroxyapatite when 5 wt.% Bioglass®45S5 is used, someregions are coveredwith glassy phasewhile the remaining regions arecrystalline. Pure hydroxyapatite and compositions with 1 and 2.5 wt.%Bioglass®45S5 have a microstructure similar to the crystalline part ofthe micrographs shown in Fig. 4(a). The grain size in all compositionswith less than 5 wt.% Bioglass®45S5 are in the range of 1–2 µm. On theother hand, the compositions with 10 and 25 wt.% Bioglass®45S5(Fig. 4(b, c and d) exhibits an amorphous glassy matrix with the

compositionwith 10 wt.% Bioglass®45S5 has crystalline Ca5(PO4)2SiO4

and the composition with 25 wt.% has Na3Ca6(PO4)5 has crystallineparticles within a glassy matrix.

In order to differentiate the mineralization behavior in thepresence of bone marrow stromal cells and in its absence, thedifferent ceramic chemistries were placed in media with and withoutbone marrow stromal cells. When placed in media without BMSCcompositions of hydroxyapatite with up to 5 wt.% Bioglass®45S5indicate some amounts of dissolution followed by reprecipitation inother regions. The dissolution results in the formation of microporosity. On the other hand compositions with Ca5(PO4)2SiO4 andNa3Ca6(PO4)5 in a silicate matrix shown in Fig. 5(a, b) formed anapatite layer with the latter composition showing the largest levels ofapatite formation. When the bioceramics were placed in media withBMSCs for a period of 6 days all samples exhibited an apatite layerformation on their surfaces. Fig. 6(a,b) shows the surfaces withcomposition of Ca5(PO4)2SiO4 and Na3Ca6(PO4)5 in a silicate matrixand it is evident that the highest levels of apatite formation are seen inthe bioceramic with Na3Ca6(PO4)5 in a silicate matrix that corre-sponds to the composition with 25 wt.% Bioglass®45S5.

Fig. 2. Calibration plot of ratio of measured integrated intensities of peaks of β-TCP andhydroxyapatite. Superimposed are the measured ratios of the integrated intensities forsintered hydroxyapatite and the compositions with up to 5 wt.% Bioglass®45S5.

Fig. 3. (a) Theoretical density of the bioceramics and the measured density of the greenand sintered bioceramics. (b) Porosity of the green and sintered bioceramics.

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3.4. Mechanical property characterization

The variation of the compressive strength of hydroxyapatite andthe bioceramics with different amounts (1, 2.5, 5, 10, and 25 wt.%) ofBioglass®45S5 addition to hydroxyapatite and sintered at 1200 °C for4 h is shown in Table 2. Compressive strength of cylindrical sampleswas measured reporting load to failure divided by the cross-sectionalarea of the samples. The average compressive strength achieved forsintered hydroxyapatite is similar or better when compared toliteratures as given in Table 3.

3.5. In vitro characterization

The inductive couple plasma (ICP) analysis for calcium, phosphorous,potassium, andmagnesium concentrations in the complete bonemediathat was taken from the original media and 6 day cell culture of BMSCon the surface of hydroxyapatite, and hydroxyapatite with 1, 2.5, 5 wt.%Bioglass®45S5 andbioceramicswith Ca5(PO4)2SiO4 andNa3Ca6(PO4)5 in

a silicate matrix are shown in Fig. 7. The most significant deviation inthe chemistry of the solution was found when the bioceramic withNa3Ca6(PO4)5 was immersed for 6 days in media containing BMSC withhigher levels of Ca and lower levels of P in the media.

The proliferation of rat BMSC on hydroxyapatite and compositionswith 1, 2.5 and 5 wt.% Bioglass®45S5 as well as bioceramics Ca5(PO4)2SiO4 and Na3Ca6(PO4)5 in a silicate matrix after 3 and 6 day cellculture are shown in Fig. 8. Pico Green DNA assay was used tocompare the DNA concentration on the bioceramic substrates thatcorrelates to the number of cells on the substrates. All Bioglass® addedHA bioceramics exhibited more cell proliferation compared to puresynthetic HA samples, in a silicate matrix both after 3 and 6 dayincubation (except the composition with Na3Ca6(PO4)5 that showed amarginal decrease after 6 days). However, One-way analysis ofvariance (ANOVA) showed statistically significant difference only inpure HA in comparison to compositions with 2.5 and 5 wt.% Bioglass®

(pb0.05) and compositions with 2.5 wt.% Bioglass® in comparison tocompositions with 10 and 25wt.% Bioglass® (pb0.05) after 3 day cell

Fig. 4. Scanning electron micrographs of as sintered bioceramics with HA and (a) and (b) 10 wt.% Bioglass®45S6 and (c) 25 wt.% Bioglass®45S5. In (a) the surface is shown and in(b) the cross-section is shown. All bioceramic compositions were sintered at 1200 °C for 4 h. The bioceramic compositions with 10 wt.% Bioglass®45S5 has Ca5(PO4)2SiO4 in a silicatematrix, and the bioceramic compositions with 25 wt.% Bioglass®45S5 is made up of Na3Ca6(PO4)5 in a silicate matrix.

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culture. The composition with Ca5(PO4)2SiO4 in a silicate matrixexhibited the maximum proliferation of the rat BMSC after 6 day cellculture showing statistical significance over all bioceramic composi-tions (pb0.05).

The Alkaline Phosphatase (ALP) activity of rat BMSC cultured onHA and bioceramic compositions with 1, 2.5 and 5 wt.% Bioglass® aswell as bioceramics with Ca5(PO4)2SiO4 and Na3Ca6(PO4)5 in a silicatematrix in complete bone media for 3 and 6 day is shown in Fig. 9. TheALP activity, which was normalized on the basis of DNA concentrationper sample, was found to be the most in the samples that containNa3Ca6(PO4)5 compared to the other compositions. The one-wayanalysis of variance (ANOVA) showed a statistically significant differ-ence between HA and bioceramic composition with Na3Ca6(PO4)5in a silicate matrix (pb0.05) after 3 day cell culture, and except forcompositions with 1 wt.% Bioglass® compared to composition with10 wt.% Bioglass® (pN0.05) and composition with 2.5 wt.% Bioglass®

compared to composition with 5 wt.% Bioglass® (pN0.05), all biocera-mic compositions have statistically significant difference between them(pb0.05).

4. Discussion

In this study bioceramicswith different amounts of hydroxyapatiteand Bioglass®45S5 were blended and sintered together. The biocera-

mic products were characterized for their structure and physical andmechanical characteristics and were examined for biocompatibilityand feasibility as substrates for differentiation and proliferation ofbone marrow stromal cells.

4.1. Structure of sintered bioceramic compositions

Powder X-ray diffraction of the sintered bioceramics indicates thatpure hydroxyapatite retains its structure after sintering with noincrease in β-TCP content. In compositions with 1 and 2.5 wt.%Bioglass®, β-TCP is the only new phase detected. On increasing thecontent of Bioglass®45S5 to 5 wt.% the amount of β-TCP increases(Fig. 2), indicating the decomposition of hydroxyapatite. Otherstudies of pure hydroxyapatite have indicated that decomposition ofhydroxyapatite occurs in the presence of moisture resulting in theformation of β-TCP and CaO at temperature above 1200 °C [32]. Onthe other hand it had also been shown that Ca deficient hydroxyap-atite when sintered at temperatures between 700 and 850 °Cdecompose to yield stoichiometric hydroxyapatite and β-TCP [33].In a study of decomposition of hydroxyapatite in an air environmentat temperatures of 1500 °C it has been shown that commercialhydroxyapatite decomposes to tetracalcium phosphate and β-TCP[34]. In thermal decomposition studies of carbonated hydroxyapatiteit has been shown that the environment plays an important role indetermining the nature of the decomposition products with wet anddry CO2 encouraging the formation of β-TCP at 1500 and 1300 °Cwhile the presence of wet and dry N2 resulting in the formation of β-TCP at 1200 and 1400 °C [35]. In a study where decomposition ofhydroxyapatite was examined in the presence of SiO2 it was shownthat a structure similar to β-TCP was formed that contained Siwithin it and is known as Si-TCP to indicate a Si-stabilized TCPwhere P in the structure is replaced by Si [36]. This structure hasbeen shown by others to be of great biological interest as it isosteoconductive [37].

There is clear evidence here that indicates that in the absence ofBioglass®45S5 there is some decomposition of hydroxyapatite onsintering at 1200 °C for 4 h but in the presence of as little as 1%hydroxyapatite up to 25% of the hydroxyapatite decomposes to formβ-TCP and the β-TCP content increases to 35% as the amount ofBioglass®45S5 is increased to 5 wt.%. The Bioglass®45S5 appears toplay the role of a trigger or nucleus that accelerates the decompositionof hydroxyapatite to β-TCP. While the presence of SiO2 is essential forthe formation of Si-TCP, the presence of P in the Bioglass®45S5precluded the formation of Si-TCP in this study and forced thedecomposition of hydroxyapatite to β-TCP.

On the other hand, in compositions with 10 and 25 wt.%Bioglass®45S5, hydroxyapatite reacts with the Bioglass®45S5 yieldingnew phases. The composition with 10 wt.% Bioglass®45S5 results inthe formation of calcium phosphate silicate (Ca5(PO4)2SiO4) and alimited amount of β-TCP, and the composition with 25 wt.%Bioglass®45S5 yields a sodium calcium phosphate (Na3Ca6(PO4)5)with no evidence of β-TCP. Bioglass®45S5 is a mixture of 45 wt.% SiO2,24.5 wt.% Na2O, 24.5 wt.% CaO, and 6 wt.% P2O5. This indicates that incomposition with hydroxyapatite that have 10 wt.% Bioglass®45S5 inaddition to the crystalline phase Ca5(PO4)2SiO4 there is an amorphousphase rich in Na, P, O with some Si present. The scanning electronmicrograph shown in Fig. 4(b) of the surface of this ceramiccomposition indicates the presence of whiskers embedded within aglassy matrix, however an image of the cross-section indicates auniform microstructure as shown in Fig. 4(c). In a closer examinationof the chemistry of the whiskers on the surface using microspotenergy dispersive spectroscopy it is evident that the composition ofthe glassy matrix and that of the whiskers is essentially the sameindicating that the whiskers are nucleating and growing on thesurface from the glassy matrix. The formation of calcium phosphatesilicate by adding Bioglass®45S5 to hydroxyapatite has been observed

Fig. 4 (continued).

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by other investigators as well [2,27]. On the other hand, in the currentstudy the composition with hydroxyapatite with 25 wt.% Bio-glass®45S5 exhibits the formation of a new phase with stoichiometryof sodium calcium phosphate (Na3Ca6(PO4)5) with the completeabsence of Si in the crystalline phases. This indicates that thecrystalline Na3Ca6(PO4)5 is embedded within an amorphous silicatematrix. The microstructure shown in Fig. 4(d) indicates that theamorphous silicate matrix is the continuous phase and is presentas a glassy phase on the surface. Bioglass®45S5 has been shown tocrystallize as Na2CaSi3O8 or Na2Ca2Si3O9 in a glassy matrix whenheated to temperatures of 1200 °C [23,38,39], however, these phasesare absent in our study indicating that prior to the formation ofcrystalline phases the amorphous bioglass reacts with hydroxyapatiteto form new chemical species.

4.2. Mechanism of new phase formation

Compositions with less than 5 wt.% Bioglass®45S5 indicateincreased levels of decomposition of hydroxyapatite with Bio-glass®45S5 content. Earlier studies on decomposition of hydroxyap-atite have indicated that the initiation of the decomposition istriggered by the migration of Ca++ ions out of the structure to form

CaO [32]. A possible reaction accounting for the decomposition ofhydroxyapatite is shown in Eq. (2).

Ca10ðPO4Þ6ðOHÞ2⟶3Ca3ðPO4Þ2 þ CaO þ H2O ð2ÞIn the presence of a silicate glass that contains P2O5 the CaO can

further react to yield additional β-TCP by the reaction shown inEq. (3).

3CaO þ ½24:5Na2O∙24:5CaO∙45SiO2∙6P2O5�⟶Ca3ðPO4Þ2þ Amorphous glassy phases ð3Þ

Fig. 6. Secondary electron scanning electron micrographs of the ceramics with(a) 10 wt.% and (b) 25 wt.% Bioglass®45S5 incubated 6 days in DMEM with bonemarrow stromal cells.

Table 2Average values of compressive strength for hydroxyapatite and five differenthydroxyapatite–Bioglass®45S5 bioceramic compositions sintered at 1200 °C for 4 h.

Composition Compressive strength (MPa)

Hydroxyapatite (HA) 64±18HA+1 wt.% Bioglass®45S5 26±2HA+2.5 wt.% Bioglass®45S5 34±5HA+5 wt.% Bioglass®45S5 79±15HA+10 wt.% Bioglass®45S5 74±8HA+25 wt.% Bioglass®45S5 131±14

Fig. 5. Secondary electron scanning electron micrographs of the bioceramic composi-tions with (a) 10 wt.% and (c) 25 wt.% Bioglass®45S5 incubated 6 days in DMEMwithout cells.

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In compositions with 10 and 25 wt.% Bioglass®45S5 additionalphases are formed by the reaction of the β-TCP with the glassy phasesin the Bioglass®45S5. A hypothetical reaction is shown for the 10 wt.%and 25 wt.% Bioglass® compositions in Eqs. (4) and (5) respectively.

Ca3ðPO4Þ2þ 0:1½24:5Na2O∙24:5CaO∙45SiO2∙6P2O5�⟶Ca3ðPO4Þ2∙2CaO∙SiO2þ Amorphous glassy Phase ð4Þ

2Ca3ðPO4Þ2þ 0:25½24:5Na2O∙24:5CaO∙45SiO2∙6P2O5�⟶2Ca3ðPO4Þ2∙Na3PO4þ Amorphous glassy Phase ð5Þ

When 10 wt.% Bioglass®45S5 is co-sintered with hydroxyapa-tite Ca++ and Si4+ cations are incorporated into the β-TCP to yieldCa5(PO4)2SiO4 (can also be written as Ca3(PO4)2∙2CaO∙SiO2). On theother hand when 25 wt.% Bioglass®45S5 is co-sintered with hydro-xyapatite Na+ cations are incorporated into the structure yieldingNa3Ca6(PO4)5 (can also be written as 2Ca3(PO4)2∙Na3PO4).

4.3. Mechanical properties of bioceramics

The hydroxyapatite–Bioglass®45S5 bioceramic compositions with1 and 2.5 wt.% displays the lowest compressive strength values while25 wt.% Bioglass®45S5 added hydroxyapatite bioceramics exhibit thehighest value indicating that high amounts of Bioglass®45S5 additionsignificantly improves the compressive strength of bioceramicsamples. All the bioceramic samples show higher compressivestrength than cancellous bone; however, in this study only compres-sive strength values achieved for 25 wt.% Bioglass®45S5 addedhydroxyapatite bioceramic sample with a crystalline Na3Ca6(PO4)5phase in a glassy silicate matrix falls within the range required forhuman cortical bone (100–230 MPa) [42–45].

4.4. In vitro bioactivity of bioceramics

In order to determine the mineralization ability and bioactivity ofeach of these sintered bioceramics in the absence of BMSCs, thesamples were immersed in DMEM for 3 and 6 days without the BMSC.

Table 3Compressive strength values for hydroxyapatite, dentine hydroxyapatite, bovinehydroxyapatite, cortical bone, and cancellous bone from other studies.

Synthetic hydroxyapatite [25,52]Sintering temperature 1140–1340 °CCompressive strength 5.26–13.81 MPaSintering temperature 1000–1300 °CCompressive strength 25.22 MPa

Dentine hydroxyapatite [53]Sintering temperature 1000–1300 °CCompressive strength 9.83–56.77 MPa

Enamel hydroxyapatite [54]Sintering temperature 1000–1300 °CCompressive strength 27.60–61.27 MPa

Bovine hydroxyapatite [53]Sintering temperature 1000–1300 °CCompressive strength 12–65 MPa

Cortical bone [42–45]Compressive strength 100–230 MPa

Cancellous bone [42,43,55]Compressive strength 2–12 MPa

Fig. 7. Inductive couple plasma (ICP) analysis for calcium, phosphorous, potassium, andmagnesium concentrations in the DMEM that was taken from the original media andmedia where samples of pure HA and bioceramics with mixtures of 1, 2.5, 5, 10, and25 wt.% Bioglass® with HA were immersed in the DMEM and BMSC and cultured for6 days.

Fig. 8. Pico Green DNA assay of rat BMSC cultured in DMEM for 3 and 6 days on thesurface of the pure HA and bioceramic compositions with different amounts ofBioglass®. The data represents mean±SD.

Fig. 9. ALP activity of the rat BMSC for 3 and 6 days measured with p-NPP assay andnormalized on the basis of DNA content per sample. Data represents mean±SD.

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All surfaces exhibited dissolution coupled with mineralization withthe composition with Na3Ca6(PO4)5 in a silicate matrix (HA–Bioglass®

bioceramic blend with 25 wt.% Bioglass® and 75 wt.% HA) exhibitingthe highest levels of mineralization, and the sintered HA without anyBioglass® showed the lowest levels of mineralization. There are someimportant distinctions in the nature of dissolution and mineralizationon the bioceramic surfaces. The pure HA surface exhibited the leastlevel of activity with the surface exhibiting some dissolution, on theother hand the composition with 1 wt.% Bioglass® exhibited a finelayer of mineralization on the surface coupled with some dissolution.The compositionwith 2.5 and 5 wt.% Bioglass® exhibits larger levels ofmineralization coupled with large dissolution of the glassy phase. Thelevel of mineralization in these three chemistries increases with theamount of β-TCP and amount of glassy phase. The composition with10 wt.% Bioglass® (Ca5(PO4)2SiO4 in a silicate matrix) exhibited veryfine deposits with a uniform layer deposited throughout. Thecomposition with 25 wt.% Bioglass® that yielded Na3Ca6(PO4)5 withina silicate matrix exhibited the largest bioactivity with extensivemineralization on the surface. This indicates that even in the absenceof BMSC the bioceramic samplewith a composition of Na3Ca6(PO4)5 ina silicate matrix exhibits significant capacity for mineralization. It isnow very well known that calcium to phosphorus molar ratio plays animportant role in the dissolution rate of calcium phosphate biocera-mics in body fluids [46]. The calcium phosphate bioceramics with aCa/P molar ratio greater than 1.67 are too stable and they are bio-inert; on the other hand, the Ca/P molar ratio lower than 1.5 increasesthe dissolution rate of the bioceramics in body fluids. Calciumphosphate silicate crystals in bioceramic compositions formed with10 wt.% Bioglass® addition have a Ca/P molar ratio of 2.5 which is toohigh and this may be the reason that this composition does not showmuch dissolution but does have mineralization. On the contrary,25 wt.% Bioglass® added compositions have a Ca/P molar ratio of 1.5which boosts the dissolution of this composition in DMEM and resultin higher mineralization [46]. In addition, studies with Bioglass® havebeen shown to exhibit faster bone production compared to HA whenimplanted in rats [47]. In these studies it has been shown that theseveral reaction layers are formed in the surface of Bioglass® togetherwith osteoblast proliferation and differentiation that explained theenhanced mineralization on the bioceramic surface [21]. Thismechanism suggests that bone bonding and bone tissue in-growthenhancement is the result of these multiple, parallel, and sequentialreactions at the bioceramic–tissue interface. In addition, it has beenshown that these interactions are related to either to physicochemicalphenomena that occur in the presence or absence of cells, or reactionsaffected by cellular activity [48].

When immersed in media with BMSC, the bioceramic composi-tions with larger amounts of Bioglass® exhibited higher levels ofmineralization compared to compositions of pure HA, and thebioceramic compositions with lower levels of Bioglass® (1, 2.5, and5 wt.% Bioglass®). This indicates that the formation of the newchemistries of calcium phosphate silicate and sodium calciumphosphate significantly influences the mineralization on the surfacein the presence of BMSC. On the other hand, all the bioceramic compo-sitions placed in DMEM without the BMSC showed dissolution onthe surface after 6 day incubation. Of all the bioceramic compositions,the 25 wt.% Bioglass® with 75 wt.% HA that yielded Na3Ca6(PO4)5 ina silicate matrix had the highest bioactivity both in the presenceand absence of BMSC. In Figs. 5(b) and 6(b) we can see extensivemineralization on the surface. The extent of mineralization in thepresence of BMSC is more than in its absence as shown by the networkstructure of apatite formation in Fig. 6(b). In an earlier study withBioglass® [21] it has been shown that in the first stages the ionsdissolve and go into solution, after which a silica gel layer is formed.This silica gel layer is believed to enhance the proliferation anddifferentiation of cells and enhances mineralization resulting in theformation of a network of apatite.

ICP analysis of the DMEM solution extracted after 6 daysimmersion of the bioceramic substrates and BMSC was conducted.The data shown in Fig. 7 indicates that there is not much of a change inthe concentration of Mg and K in all the cases. However, theconcentration of P in the solution is the lowest in the compositionswith 5, 10 and 25 wt.% Bioglass® and appears to be related to thelarger amounts of apatite deposited on the bioceramic surfaces. Inaddition, the solution in contact with the bioceramic that has acrystalline phase of Na3Ca6(PO4)5 in a silicate matrix (25 wt.%Bioglass®) shows an increase in the concentration of Ca. This indicatesthat the particles of Na3Ca6(PO4)5 embedded in a silicate matrixleaches out more Ca2+ ions into the DMEM in the presence of BMSC.However, the lower P concentration in the DMEM coupled with thesignificantly higher concentration of apatite formation on the surfaceindicates a rapid dissolution–reprecipitation process for the mineral-ization. The extent of mineralization is significantly higher in thepresence of BMSC and supports the conclusion of Tu et al. [49] whoreported that up to a certain range, higher Si concentration in thescaffolds is mitogenic for osteoblasts.

The DNA concentration in the lysate after 6 days immersion inDMEM with BMSC indicate that the scaffold with 10 wt.% Bioglass®

that contains Ca5(PO4)2SiO4 in a silicate matrix has the largestconcentration of DNA. The DNA concentration is associated withosteoblastic proliferation, and it is clearly evident that the substratethat contains Ca5(PO4)2SiO4 in a silicate matrix promotes osteoblasticproliferation better than other substrates. However, it has beenreported that extensive proliferation affects BMSC differentiationcapability and replicative potential of bioceramic substrates [50].From Fig. 9 it can be seen that bioceramics formed when 10 wt.%Bioglass® is sintered with HA (Ca5(PO4)2SiO4 in a silicate matrix) didnot show a significant amount of ALP activity. On the other hand, thelevel of ALP activity in the lysate after 6 days immersion in DMEMwith BMSC indicates that the scaffold with 25 wt.% Bioglass® thatcontains Na3Ca6(PO4)5 in a silicate matrix has the highest level ofactivity as shown in Fig. 9. ALP expression is associated withosteoblastic differentiation, and the level of ALP activity indicatesthe early stage of osteoblastic differentiation [51]. The higher ALPexpression on the Na3Ca6(PO4)5 in silicate matrix indicates that thisbioceramic composition promotes osteoblastic differentiation betterthan other compositions.

5. Conclusion

Five different ceramic compositions with different amounts ofBioglass®45S5 in synthetic hydroxyapatite (HA) were synthesized bysintering at 1200 °C for 4 h. Analysis of the structure of thebioceramics using X-ray diffraction reveals that when small amountsof Bioglass® is added (b5 wt.%) the Bioglass® performs the role of asintering aid. Higher levels of Bioglass® addition resulted in newphases formed with the bioceramic with 10 wt.% Bioglass® yielding aCa5(PO4)2SiO4 phase embedded within a silicate matrix, and thecomposition with 25 wt.% Bioglass® formed new bioceramic phaseswith a composition of Na3Ca6(PO4)5 in a silicatematrix. A compositionwith Na3Ca6(PO4)5 in a silicate matrix elicits the highest levels ofmineralization on the surface when immersed in DMEM both in thepresence and absence of bone marrow stromal cells. Alkaline Phos-phatase activity was the highest in the substrates with Na3Ca6(PO4)5in a silicate matrix indicating that there is a higher level of osteoblastdifferentiation in the presence of Na3Ca6(PO4)5 in a silicate matrix.DNA concentration that is a measure of osteoblast proliferation islargest in compositions with Ca5(PO4)2SiO4 in a silicate matrix.

Acknowledgement

The use of the Center for Characterization forMaterials and Biologyat UT Arlington is gratefully acknowledged.

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