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Comparative study on in vitro biocompatibility of synthetic octacalcium phosphate and calcium

phosphate ceramics used clinically

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2012 Biomed. Mater. 7 045020

(http://iopscience.iop.org/1748-605X/7/4/045020)

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Biomed. Mater. 7 (2012) 045020 (8pp) doi:10.1088/1748-6041/7/4/045020

Comparative study on in vitrobiocompatibility of synthetic octacalciumphosphate and calcium phosphateceramics used clinicallyShinji Morimoto, Takahisa Anada, Yoshitomo Honda1

and Osamu Suzuki2

Division of Craniofacial Function Engineering, Tohoku University Graduate of Dentistry, Sendai, Japan

E-mail: suzuki-o@m.tohoku.ac.jp

Received 6 March 2012Accepted for publication 21 May 2012Published 28 June 2012Online at stacks.iop.org/BMM/7/045020

AbstractThe present study was designed to investigate the extent to which calcium phosphate bonesubstitute materials, including osteoconductive octacalcium phosphate (OCP), displaycytotoxic and inflammatory responses based on their dissolution in vitro. Hydroxyapatite (HA)and β-tricalcium phosphate (β-TCP) ceramics, which are clinically used, as well as dicalciumphosphate dihydrate (DCPD) and synthesized OCP were compared. The materials were wellcharacterized by chemical analysis, x-ray diffraction and Fourier transform infraredspectroscopy. Calcium and phosphate ion concentrations and the pH of culture media afterimmersion of the materials were determined. The colony forming rate of Chinese hamster lungfibroblasts was estimated with extraction of the materials. Proliferation of bone marrowstromal ST-2 cells and inflammatory cytokine TNF-α production by THP-1 cells grown on thematerial-coated plates were examined. The materials had characteristics that corresponded tothose reported. DCPD was shown to dissolve the most in the culture media, with a markedincrease in phosphate ion concentration and a reduction in pH. ST-2 cells proliferated well onthe materials, with the exception of DCPD, which markedly inhibited cellular growth. Thecolony forming capacity was the lowest on DCPD, while that of the other calcium phosphateswas not altered. In contrast, TNF-α was not detected even in cells grown on DCPD, suggestingthat calcium phosphate materials are essentially non-inflammatory, while the solubility of thematerials can affect osteoblastic and fibroblastic cellular attachment. These results indicatethat OCP is biocompatible, which is similar to the materials used clinically, such as HA.Therefore, OCP could be clinically used as a biocompatible bone substitute material.

1. Introduction

Recent intensive studies have confirmed that one of the non-sintered synthetic calcium phosphate ceramics, octacalciumphosphate (OCP), displays excellent osteoconductiveproperties if implanted in various experimentally created bonedefects, including critical-sized defects, which do not heal

1 Present address: Osaka Dental University, Institute of Dental Research.2 Author to whom any correspondence should be addressed.

spontaneously throughout the lifetime of the animal [1–9].OCP has close structural similarities to hydroxyapatite (HA)and is a soluble and metastable calcium phosphate salt atphysiological pH [10], which tends to spontaneously andirreversibly convert to HA [11, 12]. One particular property ofOCP is the activation of bone formation-related cells, whereOCP enhances stromal cell differentiation into osteoblasts[8, 13–15] and osteoclast formation by raising osteoblastRANKL expression [16]. It has been proposed that the solublenature of OCP could be a determinant that induces the positive

1748-6041/12/045020+08$33.00 1 © 2012 IOP Publishing Ltd Printed in the UK & the USA

Biomed. Mater. 7 (2012) 045020 S Morimoto et al

biological responses of the ceramic [8, 17]. The dissolutionof calcium phosphate and its related materials is, however,thought to be associated with another aspect that shouldbe considered when being used in biomaterial applications;some soluble bioceramics can induce cellular toxicity[18–20], although the toxic level may be within the range thatis tolerated in vivo [21]. Therefore, analysis and elucidationof OCP characteristics regarding the possible cellular toxicand inflammatory response are of great interest for the clinicalapplication of OCP.

Biological tissue fluid is thought to have a chemicalcomposition that is saturated with respect to OCP [22, 23].Nevertheless, OCP is progressively converted to HA throughan intermediate amorphous apatite-like structure not only inphysiological neutral solution, but also in various in vivoconditions, including the subperiosteal region of cortical bone[9], bone defects [6, 8] and subcutaneous tissue [7, 24].OCP releases phosphate ions and consumes calcium ions ina one-directional manner during the progressive conversioninto HA [25, 26]. This tendency regarding the ion dissolutionmay differ from other calcium phosphates, such as dicalciumphosphate dihydrate (DCPD) [27] and β-tricalcium phosphate(β-TCP) [28], which appears to dissolve by releasing bothcalcium and phosphate ions at the same time. OCP increasesosteoblast differentiation marker genes when the cells areseeded on the crystal surfaces [13], but somehow inhibitscellular proliferation [8, 13, 26]. The capacity of OCPto enhance osteoblast differentiation increases in a dose-dependent manner [13]. It seems likely that cellular responses,including an inflammatory response, are different for thevarious types of calcium phosphate due to their intrinsicphysicochemical properties, such as solubility. Therefore, it isessential that the cytotoxic and inflammatory responses of anysubstitute material used for new bone formation clinically beat least equivalent to the materials already used in the clinicalsituation, such as HA and β-TCP.

Previous studies have reported that calcium (Ca) ions playan important role in cell adhesion [29, 30] and proliferation[31], and that inorganic phosphate (Pi) ions play multipleroles in maintaining osteoblast-like cells [32]. A physiologicalconcentration of Pi ions is a requisite for advancing bonemineralization with collagen synthesis by osteoblasts [33].In contrast, an elevation in Pi induces osteoblast-like celldeath through apoptosis [34], suggesting that the amount ofPi released from calcium phosphate ceramics by dissolutioncould be related to the cellular activity. In fact, the resorptionof Ca-deficient HA and β-TCP can be accelerated dependingon whether the crystals are sphere-like or rod-like in shape,because the dissolution may be controlled by the specificcrystal plane exposed [35, 36]. Therefore, the present studyinvestigated the cellular compatibility of OCP in terms of thesafety profile as a bone substitute material, particularly fromthe point of view of the dissolution, compared to other calciumphosphate ceramics currently used clinically.

2. Materials and methods

2.1. Calcium phosphate ceramics

Sintered HA (HOYA Co., Japan), sintered β-TCP (OlympusTerumo Biomaterials Co., Japan) and DCPD (WakoPure Chemicals Industries Ltd, Japan) were commerciallypurchased. OCP was synthesized by a precipitation methodpreviously reported [9]. All of the ceramics were ground witha pestle and mortar and sieved at 53 μm with a standard testingsieve before being used in the experiments.

The samples were analyzed by x-ray diffraction (XRD;RINT2500VHF, Rigaku Co., Japan) and Fourier transforminfrared spectrometer (FTIR; FT / IR-420, Jasco Co., Japan).The Ca/P molar ratio was determined by dissolving them inhydrochloric acid and measuring the calcium ion [Ca2+] andphosphate ion [Pi] concentrations with an inductively coupledplasma atomic emission spectrometer (ICP-AES; ICPS-8000,Shimadzu Co., Japan).

2.2. Calcium phosphate coating on microplates

Each sample was mixed with distilled water and the suspendedsolution was coated onto the inner bottom surface ofmicroplates (Corning Inc., USA). The microplates were thendried in an oven at 60 ◦C overnight.

2.3. Cell proliferation assay

The calcium phosphate-coated 48-well microplates(3 mg well−1) were prepared according to the precedingmethod (2.2). Mouse bone marrow-derived ST-2 cells(Riken, Tsukuba Science City, Japan) were cultured(3 × 104 cells/well) in 250 μL α-minimum essential medium(α-MEM; Sigma-Aldrich, USA) containing 10% fetalbovine serum (FBS; Thermo Trace, Australia) and 1%penicillin-streptomycin solution (Sigma-Aldrich, USA). Themicroplates were incubated at 37 ◦C and 5% CO2 and themedium was changed every 3–4 d during the experiment. Themedium was also changed immediately prior to performingthe assay. The number of live cells was then quantitativelymeasured at 5 h and 3, 7, 14 and 21 d using a WST-8assay (Cell Counting Kit-8, Dojindo, Japan) according to themanufacturer’s instructions.

2.4. Measurements of [Ca2+] and [Pi] and medium pH

The calcium phosphate-coated 48-well microplates(3 mg well−1) were prepared according to the precedingmethod (2.2). The α-MEM medium (10% FBS; 1% penicillin-streptomycin solution) was added to each well (250 μLwell−1) and the microplates were incubated at 37 ◦C and 5%CO2. [Ca2+] and [Pi] of the medium were determined byICP-AES. Medium pH was measured with a pH meter (twinpH B-212, Horiba, Japan). Measurements were taken at 0, 6,12, 24, 48 and 72 h after the initiation of the experiment.

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Biomed. Mater. 7 (2012) 045020 S Morimoto et al

2.5. Determination of the degree of supersaturation in themedium supernatant

The degree of supersaturation (DS) in the medium wascalculated in order to estimate the dissolution of OCP andother calcium phosphate ceramics with respect to the phasesHA, OCP, and DCPD using equation (1).

DS = IP/Ksp, (1)

where IP is ionic activity product and Ksp is solubility product.The DS value will equal one if the medium supernatant

reaches the saturated condition with respect to each calciumphosphate. The DS value is usually calculated using theanalytical results of [Ca], [Mg], [Na], [K], [P], [Cl] and [F]as well as the pH value in conjunction with the three massbalance equations for [Ca], [P] and [Mg], according to previousreports [37–39]. The calculation also assumed the presence ofHCO3

− in the fluid. The ion pairs considered were CaH2PO4+,

CaHPO40, MgHPO4

0, CaHCO3+, and MgHCO3

+. The DS wasdefined in terms of the mean ionic activity product with respectto OCP, HA and DCPD. In the present study, the DS value wascalculated based on the pH value as well as the calcium andphosphate concentrations in the supernatant solution. An ionicstrength of 150 mM of Na+ was assumed. A pH value of 7.4and physiologic partial pressure of 1.86% in carbonate wereused for the calculation. Other species, such as Mg2+ andF−, were assumed to be approximately zero. The solubilityproduct constants used were 2.63 × 10−60 for HA [40] and1.05 × 10−47 for OCP [27].

2.6. Cell toxicity assay

Each calcium phosphate sample was mixed in a 50 mlcentrifuge tube by adding α-MEM (Life TechnologiesCorp., USA) containing 5% FBS (Thermo Fisher ScientificInc., USA), 1% penicillin-streptomycin (Life TechnologiesCorp., USA), 1 mM sodium pyruvate, and 26 mM sodiumcarbonate at a ratio of 0.1 mg ml−1. The centrifuge tubes wereplaced in an incubator at 37 ◦C and 5% CO2 for 24 h. Thesolvent was then collected as an extract by filtering througha sterilizing filter (Steriflip, Millipore, USA). The extractconcentrations were adjusted by diluting with the α-MEMmedium.

Chinese hamster lung fibroblast V79 cells (JCRBcell bank, Japan) were seeded in 6-well microplates(1 × 102 cells/well) and incubated at 37 ◦C and 5% CO2 for24 h. The medium was then changed to the adjustedconcentration extract and cultured for an additional 4 d. Theextract was then changed to methanol for fixation and giemzasolution was added for staining. The number of colonies, whichwere defined as a cluster consisting of over 50 cells, werecounted. The relative colony forming rate (%) was calculatedas the ratio of the number of colonies in each extract to thenumber of colonies in the 0% extract. The IC50 (%) value,which was defined as the concentration of extract that providesa 50% relative colony forming rate, was calculated to evaluatethe cytotoxicity of the samples [41].

0 10 20 30 40 50 60

2θ (°)

(a)

(b)

(c)

(d )

*

*

Figure 1. XRD pattern of (a) DCPD, (b) OCP, (c) β-TCP and(d) HA. Asterisk indicates specific peaks of OCP.

2.7. Estimation of inflammatory response by THP-1 cells

The calcium phosphate-coated 24-well microplates wereprepared according to the preceding method (2.2). The coatingamount was adjusted to 0, 3, 6 and 9 mg per well. The humanacute monocytic leukemia cell line THP-1 (Tsukuba ScienceCity, Japan) was cultured (1 × 106 cells/well) in RPMI 1640medium (1 ml: Nacalai Tesque Inc., Japan) containing 10%FBS and 1% penicillin-streptomycin. For the positive control,10 μL of NiSO4 solution (17 μg μL−1) was added. Themicroplates were incubated at 37 ◦C and 5% CO2 for 2 d.After the incubation period, the number of cells (Cell CountingKit-8) and the concentration of TNF-α (TNF-α Human BiotrakEasy Elisa, GE Healthcare UK Ltd, UK) were quantitativelymeasured. The relative cell number was calculated by dividingthe number of cells by the control well.

2.8. Statistical analysis

Results were shown as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was carried out to comparegroup means (SAS Enterprise Guide 4.2, SAS Institute, Inc.).If the difference was significant, a Tukey multiple comparisonanalysis was carried out as a post-hoc test.

3. Results

3.1. Characterization of the calcium phosphates

Figures 1 and 2 show the XRD pattern and FTIR spectra ofthe samples. The patterns and spectra corresponded to thoseof calcium phosphates which had been previously reported[8, 42]. Specific peaks for OCP were observed in the XRDshown in figure 1 (2θ = 4.7◦ and 33.6◦) and FTIR in figure 2(1105, 1074, 1039 and 1024 cm −1). The Ca/P molar ratio ofthe samples was close to each stoichiometric composition ofcalcium phosphate (see table 1).

3.2. Cell proliferation on the calcium phosphate-coatedmicroplates

Figure 3 shows the number of proliferating ST-2 cells on thecalcium phosphate-coated microplates. Very few living cellswere detected on the DCPD-coated plates after 3 d of culture.

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Biomed. Mater. 7 (2012) 045020 S Morimoto et al

50070090011001300

Wavenumber (cm -1)

(a)

(b)

(c)

(d)

* ***

Figure 2. FTIR spectra of (a) DCPD, (b) OCP, (c) β-TCP and(d) HA. Asterisks indicate specific peaks of OCP.

0

5

10

15

0 7 14 21Culture time (days)

Cel

l num

ber

(×10

4 )

DCPD OCP β-TCP HA

Figure 3. Cell proliferation of ST-2 cells on calciumphosphate-coated plates. Open diamond: DCPD; open circle: OCP;open triangle: β-TCP and open square: HA.

Table 1. Chemical composition and Ca/P molar ratio of calciumphosphate ceramics used.

Ca P Ca/P Ca/PSamples (wt%) (wt%) Molar ratio Theoretical value

DCPD 24.1 17.8 1.05 1.00OCP 34.6 20.1 1.33 1.33β-TCP 41.7 21.1 1.53 1.50HA 43.5 20.2 1.66 1.67

Although the cell number was relatively low on the OCP-coated plates after 5 h of incubation, the number increasedover time and became similar to those grown on the othercalcium phosphates.

3.3. Medium [Ca2+] and [Pi] and pH of the calciumphosphate-coated microplates

Figures 4 and 5 show the medium [Ca2+] and [Pi] afterincubation with calcium phosphates. The [Ca2+] in the medium

0

1

2

3

0 24 48 72

Incubation time (hours)

[Ca 2+

] (m

M)

DCPD OCP β-TCP HAcontrol

Figure 4. The time course of Ca2+ concentration in the cell culturemedium. Open diamond: DCPD; open circle: OCP; open triangle:β-TCP; and open square: HA; cross: control (without calciumphosphates).

0

5

10

15

20

0 24 48 72

Incubation time (hours)

[Pi]

(m

M)

DCPD OCP β-TCP HAcontrol

Figure 5. The time course of Pi concentration in the cell culturemedium. Open diamond: DCPD; open circle: OCP; open triangle:β-TCP; and open square: HA; cross: control (without calciumphosphates).

from OCP-coated plates decreased over time and remained atrelatively low levels. The [Pi] in the medium from DCPD-coated plates gradually increased to 16.2 mM after 72 h ofculture. In addition, the pH of the medium in the DCPD-coatedplates gradually decreased to 6.1 after 72 h (see figure 6).

3.4. DS value in the medium supernatant

The DS values are shown in table 2. The DS of DCPD becamesaturated within 72 h of culture. The DS values of the mediafrom the OCP, β-TCP and HA-coated plates all remainedundersaturated with respect to DCPD, highly supersaturatedwith respect to HA, and supersaturated with respect to OCP.

3.5. Cytotoxicity of DCPD

The IC50 values for OCP, β-TCP, and HA were over 100%(see figure 7), which suggested that these calcium phosphate

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Biomed. Mater. 7 (2012) 045020 S Morimoto et al

Table 2. Degree of supersaturation (DS) of the media after incubation of calcium phosphate ceramics.

DS at 6 h DS at 72 h

Samples HA OCP DCPD HA OCP DCPD

Control 2.60 × 1012 3.78 × 103 6.43 × 10−1 8.78 × 1011 2.74 × 103 7.45 × 10−1

DCPD 5.84 × 1012 9.94 × 103 9.35 × 10−1 4.91 × 108 1.75 × 103 6.69OCP 2.37 × 1010 5.05 × 101 1.73 × 10−1 3.15 × 1010 7.53 × 101 2.06 × 10−1

β-TCP 1.92 × 1012 1.56 × 103 3.93 × 10−1 1.42 × 1012 8.75 × 102 2.96 × 10−1

HA 1.33 × 1012 1.56 × 103 4.45 × 10−1 5.61 × 1011 5.48 × 102 2.96 × 10−1

5

6

7

8

9

0 24 48 72Incubation time (hours)

pH

DCPD OCP β-TCP HAcontrol

Figure 6. The time course of pH in the cell culture medium. Opendiamond: DCPD; open circle: OCP; open triangle: β-TCP; and opensquare: HA; cross: control (without calcium phosphates).

0 25 50 75 100Extract concentration (%)

50

100

Rel

ativ

e co

lony

form

ing

rate

(%

)

OCP β-TCP HADCPD

Figure 7. Relative colony forming rate of V-79 cells in the extractmedium after 4 d. Open diamond: DCPD; open circle: OCP; opentriangle: β-TCP and open square: HA.

ceramics are not cytotoxic in nature. On the other hand, theIC50 value for DCPD was 87%, suggesting that DCPD isweakly cytotoxic.

0

50

100

150

3 mg 6 mg 9 mg positive controlCoating amount (mg)

DCPD OCP β-TCP HA

Rel

ativ

e ce

ll nu

mbe

r(%

)

*

***

*

* ***

Figure 8. Relative cell number of THP-1 cells cultured in thecalcium phosphate-coated plates for 2 d. Data represent the mean ±s.d. (n = 3 per group). ∗: P < 0.05.

0

1

2

0 mg 3 mg 6 mg 9 mg positivecontrolCoating amount (mg)

N.D. N.D. N.D.N.D.

DCPD OCP β-TCP HA

TN

F-α

/ cel

l(ng

)

(DCPD, OCP, β-TCP, HA)

Figure 9. TNF-α secretion from THP-1 cells cultured on calciumphosphate-coated plates for 2 d. The amount of TNF-α isnormalized by the number of live cells. Data represent the mean ±s.d. (n = 3 per group). ∗: P < 0.05.

3.6. TNF-α secretion in THP-1 cells treated with OCP

OCP showed a relatively small cell number at 9 mg coating(figure 8). Figure 9 shows the amount of secreted TNF-α perlive cell induced by calcium phosphate ceramics. TNF-α wasonly detected in the positive control, but was not detectable inwells with cells grown in the presence of the calcium phosphateceramics.

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Biomed. Mater. 7 (2012) 045020 S Morimoto et al

4. Discussion

The present study provides evidence that the cytocompatibilityof OCP is similar to other calcium phosphates that are usedclinically to repair bone defects. Calcium phosphate ceramics,especially HA and β-TCP, have been well established asmaterials that are biocompatible [43, 44]. In contrast, it hasbeen reported that calcium phosphates such as DCPD-basedcement, which has relatively higher solubility than HA underphysiologic pH and therefore affects the environmental pHaround the surface, can have some effects on cells [20,45]. Based on pathological calcification and the experimentalfindings of implantation or in vitro cell responses, somecalcium phosphates, such as biphasic calcium phosphateceramics, evoke an inflammatory response from synovialcells [46]. Thus, some reports have shown that calciumphosphate-dependent inflammatory responses can be induced.The present study analyzed the cellular compatibility ofcalcium phosphates with regard to the crystal chemistry, andparticularly the dissolution of calcium phosphates, includingOCP.

A series of calcium phosphates that had closestoichiometric composition was assessed in this study. Itis known that HA displays a wide range of Ca/P molarratios from 1.5 to 1.67 [47]. HAs having Ca/P molar ratiobelow 1.67 are recognized as Ca-deficient HA [47]. HAcan exhibit different dissolution behavior with tissue andcells depending on the stoichiometry (Ca/P molar ratio andinclusion of impurities, such as carbonate, magnesium andfluoride ions), the crystallinity and the crystal morphology[48–50]. A non-stoichiometric composition of OCP hasalso been reported [6, 17, 51, 52]. The results of acrystallographic study suggested that an OCP structure withexcess hydrogen would have a non-stoichiometric chemicalformula, Ca16H4+X(PO4)12(OH)X · (10–X)H2O, which has aneven closer resemblance to the structure of HA than previouslyanticipated [51]. We have previously shown that non-stoichiometric OCP exhibits a Ca deficiency and containsexcessive acid phosphate in the structure [6, 17, 52, 53]. Anon-stoichiometric OCP that has a Ca/P molar ratio (Ca/P1.37) larger than the stoichiometric OCP (Ca/P 1.33), whichis obtained by hydrolysis in hot water, is capable of increasingthe bone regenerative properties more than that by Ca-deficientOCP (Ca/P 1.26) if implanted in the rat tibia intramedullarycanal [6].

The dissolution of calcium phosphates can be evaluatedby DS values in the medium supernatant after thecrystal incubation by determining calcium and phosphateconcentrations, pH, and ionic strength, assuming the presenceof ion pairs, such as MgHCO3 [37–39]. It was apparent that thefresh medium had a composition that was supersaturated withrespect to the phases of HA and OCP, but undersaturated withrespect to the phases of DCPD (see table 2). The results showedthat the medium after DCPD incubation for 72 h becamesaturated with respect to the phases of DCPD. The DS valuewith respect to the phase of HA was 1788 times lower than thevalue in fresh medium. The fact that the DS at 6 h in DCPD wasundersaturated with respect to DCPD suggests that the change

in the DS value may increase the potential precipitation of HA[37–39]. The dissolution of DCPD induces a decrease in pHto approximately 6, which can induce cytotoxic effects (underpH = 6.4) [54]. Although the release of Pi was also observedfor other calcium phosphates, the finding of a markedly higherlevel of DCPD and the fact that the pH did not decrease inthe presence of other calcium phosphates suggest that the pHchange during the DCPD incubation may be due to the Pirelease caused by the dissolution. After 72 h of incubation, theDS of β-TCP (1.42 × 1012) was the highest, followed by HA(5.61 × 1011) and OCP (3.15 × 1010), respectively, with respectto the phase of HA. DCPD had the lowest DS (4.91 × 108)with respect to the phase of HA. The potential capability ofHA formation was 2892 times higher in β-TCP, 1142 timeshigher in HA, and 64 times higher in OCP than DCPD. Ahigher DS value does not always indicate HA formation [37–39]. The rate of HA crystal nucleation is critical for the phasetransformation. However, previous studies have confirmed thatOCP can be converted to HA in similar culture conditions asused in this study [8, 13].

Incubation with OCP for 5 h induced the greatestinhibition of initial attachment of ST-2 cells onto coatedplates. The reason could be explained by the lowest calciumconcentration in the medium. The calcium concentration playsan important role in cell adhesion [29, 30]. In contrast, theproliferation of ST-2 cells on DCPD was markedly inhibitedafter 3 d of culture, and the growth did not occur even afterthe prolonged incubation. This may be an effect of excessiveconcentration of Pi in the medium from 24 h (over 5 mM), aconcentration recognized to lead to cell apoptosis [34]. Theobservation that the cells initially attached onto DCPD butcould not subsequently proliferate was most likely due to thephysicochemical change (the fast dissolution). HA and β-TCPwere the most suitable scaffold for cell proliferation. However,OCP somewhat inhibited cell proliferation compared to HAand β-TCP. The inhibitory effect of OCP on osteoblastic cellproliferation was also observed in previous studies comparedthat by to various forms of HA, including non-sinteredstoichiometric HA, a non-stoichiometric Ca-deficient HA(Ca/P 1.50) obtained through direct precipitation, and a Ca-deficient HA (Ca/P 1.46) obtained from OCP by hydrolysisin hot water [8, 26]. The proliferation of cells grown on OCPwas similar to the cells grown on HA and β-TCP after theprolonged incubation. Osteoblastic ST-2 cells grown on HAand β-TCP had better attachment and proliferation properties,suggesting that HA formation is critical for providing bettercellular attachment characteristics [8, 13, 26, 55].

The IC50 value of DCPD was 87% based on fibroblasticgrowth and colony formation, supporting the finding thatDCPD had an inhibitory effect on ST-2 cell attachment andproliferation. Increasing the extract concentration of DCPDmay cause an increase in excessive Pi, which can inhibitthe cellular activity. On the other hand, the IC50 valuesof HA, β-TCP and OCP were over 100%. The extractionexperiment was conducted for a short period of time comparedto the cellular proliferation studies. Therefore, although theconversion from OCP to HA may not advance completelyin the extraction, it seems likely that the cytotoxic effects

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Biomed. Mater. 7 (2012) 045020 S Morimoto et al

of OCP should be very minimal, if at all. It is conceivablethat OCP has similar cytotoxic effects as HA and β-TCPif estimated using the standardized testing method [41]. Ithas been shown that the RAW264.7 macrophage cell linecan form osteoclasts when in contact with various DCPDcements with distinct solubilities and in the presence ofthe receptor for nuclear kappa B ligand (RANKL) [20].This study suggests that cell-mediated biodegradation canbe controlled by the soluble nature of DCPD. In contrast,the present results showed that no inflammatory responsewas induced by any calcium phosphate ceramics, includingDCPD or OCP, if the THP-1 cells were inoculated with theircoatings. THP-1 cells are classified as a non-adhesive celltype in nature. However, the cells were most likely in frequentcontact with calcium phosphate ceramics under the presentcoating conditions. Together, these results suggest that calciumphosphate ceramics are biocompatible, even in DCPD, but thatthe ionic condition around these ceramics can affect cellularactivity, especially the cellular attachment of osteoblasticcells. The physicochemical environment induced by calciumphosphate ceramics, depending on the crystal phase, could bea factor that determines the level of cellular activity. The factthat ST-2 cell proliferation on OCP was inhibited in the earlystages of the culture but reached a similar level as the cellsgrown on HA for a prolonged period of time, supports thisconcept. Therefore, OCP has a comparable biocompatibilityto HA and β-TCP.

5. Conclusions

This study investigated the possible use of OCP as animplant material by comparing the cytotoxic and inflammatoryresponses with a series of calcium phosphate ceramics,including HA and β-TCP, which are widely used as bonesubstitute materials for bone repair. The results confirmedthat the physicochemical dissolution of calcium phosphateceramics may play a role in controlling the cytotoxicity, asestimated by osteoblastic and fibroblastic cells. OCP exhibiteda biocompatability as well as bioceramics used clinically,including HA and β-TCP. Therefore, we hypothesize thatsynthetic OCP, which has been confirmed to have excellentosteoconductivities in various animal experiments, can beapplied to many clinical situations in the field of orthopedic,dental and oral surgeries based on the biocompatibility.

Acknowledgment

This study was supported in part by grants in aid (17076001,19390490, 23390450, 23659909 and 23106010) from theMinistry of Education, Science, Sports and Culture of Japan.

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