Effects of exogenous phosphorus and silicon on osteoblast differentiation at the interface with...

Effects of exogenous phosphorus and silicon on osteoblastdifferentiation at the interface with bioactive ceramics

Gautam Gupta,1 Sreenatha Kirakodu,2 Ahmed El-Ghannam3

1Biomet Inc., 56 E Bell Drive, Warsaw, Indiana 465822Center for Oral Health Research, College of Dentistry, University of Kentucky, Lexington, Kentucky 405063Department of Mechanical Engineering and Engineering Science, University of North Carolina at Charlotte,

Charlotte, North Carolina 28223

Received 22 December 2009; revised 13 April 2010; accepted 21 May 2010

Published online 15 September 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.32915

Abstract: In this study, we have investigated the effects of

dissolved phosphorus and silicon on osteoblast differentia-

tion in vitro. Neonatal rat calvarial osteoblasts were seeded

on silica–calcium phosphate composites (SCPCS), hydroxyap-

atite (HA-200), and tissue culture polystyrene (TCPS) and

incubated over 4 days in media containing 0 {minimal essen-

tial medium [MEM] (�)} or 3 mM b-glycerophosphate [MEM

(þ)]. Inductively coupled plasma analysis showed that P-con-

tent in original MEM (þ) was 225% higher than that in MEM

(�). Moreover, P-content in MEM (þ) significantly increased

to 3.4–4.4 mM and 3.6–4.7 mM after 2 and 4 days incubation

with SCPC, respectively, owing to material dissolution and

exogenous phosphate supplementation. In contrast, P-con-

tent in MEM (þ) showed no change upon incubation with HA

or TCPS. The P-content in MEM (�) incubated with SCPC was

considerably lower than that in MEM (þ). SCPC exhibited

controlled Si-release in cell culture media [MEM (�) or MEM

(þ)], with Si-rich SCPC showing a significantly greater disso-

lution than Si-poor SCPC. Moreover, SCPC, unlike HA, dem-

onstrated a cell- and solution-mediated dissolution over 4 days.

Quantitative real-time PCR showed that in MEM (�), osteocal-

cin and osteopontin mRNA expression on Si-rich SCPC was

significantly greater than that on HA, suggesting that Si plays

an important role in enhancing bone-cell differentiation. How-

ever, osteoblast phenotypic expression on SCPC was signifi-

cantly decreased after 4 days incubation in MEM (þ), indicating

that sustained exposure to elevated P-levels in the media can

downregulate osteoblast function. Our results demonstrate that

the controlled dissolution of SCPC provides a natural stimulus

for bone-cell differentiation in vitro and could obviate the need

of exogenous phosphate supplementation. VC 2010 Wiley Periodi-

cals, Inc. J Biomed Mater Res Part A: 95A: 882–890, 2010.

Key Words: silica–calcium phosphate composite, ionic disso-

lution, phosphate supplementation, osteoblast gene expres-

sion, real-time PCR

INTRODUCTION

The dissolution behavior of bioceramics in physiological sol-utions and its effect on the cellular response is of both fun-damental and clinical importance. Material dissolution leadsto graft resorption, which is a critical requirement for tissueregeneration at the implant site.1–3 Moreover, ionic dissolu-tion from the material is responsible for implant bioactivityand bone-bonding ability through the development of a bio-logical apatite surface layer.1,4,5

Degradation of the implant material depends on thephysicochemical properties like chemical and phase com-position, porosity, and matrix ultrastructure.6,7 Moreover,graft-resorption in vivo and in vitro is both a cell- and solu-tion-mediated phenomenon.8,9 Cellular activity affects the dis-solution reactions at the tissue-implant interface.8 However,the implant degradation rate can in turn affect the bone-cellfunction. Materials with low-dissolution rates, like stoichio-metric hydroxyapatite (HA), exhibit restricted bioactivityand bone integration.1,10 On the other hand, b-tricalciumphosphate is characterized by a relatively high-dissolution

rate that can affect the implant stability and could lead toan immunological response.11 However, controlled release ofionic products from material dissolution can in fact stimu-late and enhance bone-cell function. It was observed thathuman osteoblasts cultured in media treated with ionic dis-solution products from bioactive glass (BG) exhibited signifi-cantly upregulated IGF-II gene expression and protein syn-thesis compared to the cells cultured in untreated controlmedia.12 Moreover, dissolved Si has been shown to increasecollagen-I synthesis and enhance osteoblast differentiation.13

Furthermore, it has been suggested that dissolved silicaincreases the bioactivity of the bioceramics.14 In addition,dissolved Ca and P ions have been shown to affect osteo-blast function in a positive or negative manner dependingupon the concentration of these ions in the media. Exposureto Ca2þ was observed to enhance osteoblast function.15,16 Inanother study, phosphate in the media was shown to act asa specific signal for the induction of osteopontin geneexpression and eventually protein synthesis by osteoblast-like cells.17–19 However, it has also been reported that

Correspondence to: A. El-Ghannam; e-mail: [email protected]

Contract grant sponsor: Culpeper; contract grant number: 03-177

882 VC 2010 WILEY PERIODICALS, INC.

exposure to elevated phosphate concentration can activateosteoblast apoptosis by profound loss of mitochondrial mem-brane potential. When osteoblast-like cells from humanexplants were exposed to an inorganic phosphate (Pi) con-centration of 1–7 mM, there was a time- and dose-dependentdecrease in the cell viability.20 Moreover, it was demonstratedthat Ca2þ modulated Pi-induced osteoblast apoptosis.21,22

To promote mineral deposition, 3–10 mM of b-glycero-phosphate (b-GP) is routinely used by researchers conduct-ing in vitro experiments with osteoblast-like cells. However,the effects of exogenous phosphate supplementation havebeen debated in the recent years. On the one hand, b-GPaddition has been shown to support mineral depositionresembling woven bone.23 On the other hand, elevatedCa/PO4 ionic products in culture media led to ectopic min-eral deposition, whenever 2 mM b-GP concentration levelwas exceeded.24 Moreover, it has been suggested that b-GPalters the phosphorylation status of casein kinase II, anextracellular enzyme involved in mineralization, leading to adysregulation of the mineralization process, with abnormalcalcification and excessive crystal growth.25 However, itwould be prudent to realize that direct comparison betweenthese studies is not feasible due to the different experimen-tal conditions involved.

Silica–calcium phosphate composite (SCPC) is a novelporous bioceramic that is characterized by superior bioac-tivity and resorbability than HA or BG.26,27 Si-rich SCPCcompositions developed biological surface apatite layer onlyafter 2 h of immersion in simulated body fluids. In contrast,a similar layer was detected on BG surface only after 8 daysunder the same experimental conditions. Moreover, the dis-solution rate of SCPC was demonstrated to be significantlygreater than that of BG.26 Mesenchymal bone marrow pluri-potent cells attached to SCPC produced mineralized extrac-ellular matrix and bonelike tissue, whereas cells attached toHA-200 produced limited unmineralized extracellular ma-trix.27 The enhanced bioactivity and resorbability of SCPCwas correlated to its chemical composition and nanophaseultrastructure. In this study, we have investigated the effectsof dissolved phosphorus and silicon ions, in the presence orabsence of exogenous phosphate supplementation, on osteo-blast differentiation in vitro. Quantitative real-time PCR wasused to analyze the gene expression of neonatal rat calvarialosteoblasts attached to SCPC composites when they were cul-tured in the presence or absence of b-GP supplementation.

MATERIALS AND METHODS

Composite preparationSCPC10, SCPC30, and SCPC50, with initial Si/CaP ratio(wt %) 10/90, 30/70, and 50/50, respectively, were pre-pared as reported elsewhere.28 The original powders weremixed in polyethylene bottles over a roller for 24 h, ther-mally treated at 800�C (Thermolyne 30400, BarnsteadInternational, Dubuque, IA) for 1 h and then ground to sizerange 45–150 lm. Porous discs were then prepared by mix-ing SCPC ceramic particles (45–150 lm) with 40 vol %polyethylene glycol (PEG, Sigma, St. Louis, MO) particulateswith particle-size equally distributed in 90–250 lm and

250–425-lm range. The SCPC–PEG blend was mixed over aroller for 24 h and then compressed at room temperatureinto cylinders (diameter 13 mm, height 2 mm) under a uni-axial load of 160 MPa. The samples were then heated at350�C for 24 h to remove the polymer and then sintered at800�C for 1 h in a furnace. Transmission electron micros-copy showed that SCPC composite was made up of crystalsin the size range 50–300 nm.27 Porosity analyses using mer-cury intrusion porosimetry, scanning electron microscopy,and high-resolution X-ray computed tomography revealedthat the SCPC scaffolds were characterized by a homoge-nously distributed interconnected network of pores in awide size range (3 nm–650 lm).28 Moreover, porosimetryanalysis had previously showed that the total porosity ofSCPC scaffolds was between 32 and 40%.28 It should benoted that this is the originally engineered porosity of scaf-folds made with the resorbable SCPC material. As the SCPCmaterial resorbs in vivo, the overall porosity of scaffold willincrease, facilitating new bone ingrowth in the scaffolds.

Cell culturePorous SCPC discs as prepared earlier were used for thecell-culture experiments. Porous coralline HA discs (HA-200from Interpore Cross, Irvine, CA) containing about 50%porosity were used as a positive control, while tissue cul-ture polystyrene (TCPS) was used as a negative control forthe gene-expression analysis. SCPC and HA discs were steri-lized by immersing them in 70% ethanol for 15 min. Neona-tal rat calvarial osteoblasts were enzymatically isolatedusing the collagenase digestion technique described previ-ously.29 Briefly, the calvarias of 1–2-day-old rat pups wereexcised, stripped of soft tissue, and subjected to a series of20-min digestions with a collagenase–trypsin solution. Thecells from the first two digestions were discarded. The cellsfrom each successive digestion were suspended in separateflasks in a-minimal essential medium (a-MEM, Gibco, Invi-trogen Corp., Carlsbad, CA) supplemented with 10% fetalbovine serum (Gibco), 0.1% penicillin–streptomycin (Gibco),and 0.06% fungizone (Gibco) and incubated at 37�C under5% CO2 and 95% humidified air. After the cells became con-fluent, they were trypsinized, centrifuged, and homoge-nously suspended in 2 mL of MEM. Aliquots of the cell sus-pension containing 5 � 105 cells were pipetted on thesample surface and incubated for 2 and 4 days in 6-mL tis-sue culture media. The volume of tissue culture media wasselected such that its buffering capacity was not exceededfrom biomaterial dissolution products, thereby maintainingthe pH at around 7.0 for the entire duration of the study.For each time point, one set of samples were incubated inMEM containing 50 lg/mL ascorbic acid, 3 mM b-GP, and10�8M dexamethasone; whereas the other set of sampleswere incubated in MEM containing 50 lg/mL ascorbic acidand 10�8M dexamethasone but no b-GP. For the 4-day timeexperiment, the culture media were exchanged after every 2days. The media from 2 and 4 days experiments were col-lected and stored in 15-mL plastic centrifuge tubes at �20�Cfor ionic concentration analyses. A parallel experiment, underthe same conditions, was conducted with SCPC and HA discs

ORIGINAL ARTICLE

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | 1 DEC 2010 VOL 95A, ISSUE 3 883

that were not seeded with cells to compare the dissolutionbehavior of these bioceramics in the presence and absenceof cells. Six replicates of each type of sample were used inthe gene expression experiments. The neonatal rat calvarialcells have been repeatedly shown to acquire osteoblastcharacteristics in vitro as indicated by the high-alkalinephoshpatase activity, the mRNA expression of osteocalcin,osteopontin, and bone sialoprotein, and deposition of miner-alized extracellular matrix and calcified nodules.29,30 Addi-tionally, osteoblast-like cells have been known to demon-strate osteoblast phenotypic gene expression on calciumphosphate and Si-containing calcium phosphate materials asearly as 3 days.31 Thus, for this investigation, early timepoints of 2 and 4 days were selected to capture the effect ofSCPC chemical composition on the osteoblast phenotypicexpression of the neonatal rat calvarial cells.

Quantitative real-time PCRAfter 2 and 4 days in culture, the discs were removed to afresh culture plate, the cells were lysed, and RNA extractedusing TRIZOL reagent (Invitrogen, Carlsbad, CA). The geneexpression of cells attached to SCPC, HA, and TCPS sampleswere analyzed using techniques described previously.28 Theexpression levels of the target genes were calculated by nor-malizing the mRNA for a particular gene against the mRNAof the housekeeping gene b-actin. The efficiency correctionsfor the PCR reactions were performed by relating thethreshold cycle values obtained from the samples to anexternal standard curve. Genes for the real-time PCR wereobtained from rat and included collagen-I (F:50-ACCCCAAGGAGAAAAAGCAT-30; R:50-ATGTCCATTCCGAATTCCTG-30) osteo-calcin (F:50-GCATTCTGCCTCTCTGACCT-30; R:50-CTAAACGGTGGTGCCATAGA-30), osteopontin (F:50-ACAGTCGATGTCCCTGACG-30; R:50-TGAAACTCGTGGCTCTGATG-30), osteonectin(F:50-AAACATGGCAAGGTGTGTGA-30; R:50-CCAGTGGACAGGGAAGATGT-30), and b-actin (F:50-GCTACAGCTTCACCACCACA-30;R:50-CAGTGAGGCCAGGATAGAGC-30). The primers weredesigned using Primer-3 software (MIT, Cambridge, MA),and the HPLC purified primers for these genes wereobtained from Integrated DNA Technologies (Coralville, IA).

Ionic concentration analysisThe concentrations of Ca, P, and Si ions in the media sam-ples collected from 2 and 4-day experiments were evaluatedusing inductively coupled plasma-optical emission spectrom-eter (ICP–OES) (Optima DV 2000, PerkinElmer, Wellesley,MA). The ICP machine was operated under conditionsreported previously.29

Statistical analysisThe ionic concentration analyses were conducted with n ¼6. For the gene expression analysis, data from three cDNAamplifications were combined for each sample (n ¼ 3). Allthe data have been expressed as mean 6 SD, and were ana-lyzed using Sigma Stat software (SPSS, Chicago, IL). Thedata were evaluated with one-way ANOVA after the datapassed normality and equal variance tests. All pair-wise

multiple comparisons were conducted with the Tukeymethod. Statistical significance was considered at p < 0.05.

RESULTS

Ionic concentration analysisCa concentration. Figure 1(a,b) shows the ICP analysis ofCa2þ concentration in MEM (�) and MEM (þ) after 2 and 4days of incubation with SCPC-, HA-, or TCPS-cell systems.After 2 days, MEM (�) incubated with SCPC30 exhibited asignificant increase in Ca2þ concentration (75 ppm) com-pared to the original MEM (�) solution (TCM, 66 ppm). Onthe other hand, MEM (�) incubated with HA showed a sig-nificant decrease in Ca2þ concentration (44 ppm). Ca2þ con-centration in MEM (þ) incubated with SCPC10, SCPC30, orSCPC50 was 77 ppm, 74 ppm, and 79 ppm, respectively,which was significantly higher than the control TCM. On theother hand, MEM (þ) incubated with HA showed a signifi-cant decrease in Ca2þ concentration (40 ppm) after 2 daysof incubation. After 4 days, the Ca2þ concentration in MEM(�) incubated with SCPC30 and SCPC50 was 76 ppm and75 ppm, respectively, which was significantly higher thanthe control TCM. The MEM (�) incubated with HA showeda significant decrease (54 ppm) in Ca2þ concentration.Moreover, MEM (þ) incubated with SCPC50 ([Ca] ¼ 97ppm) or SCPC30 ([Ca] ¼ 76 ppm) showed a significantincrease in Ca2þ concentration after 4 days, while that incu-bated with HA ([Ca] ¼ 54 ppm) showed a significantdecrease. It was also observed that the Ca2þ concentrationin culture media [MEM (�) or MEM (þ)] after 2 and 4 daysof incubation with SCPC50 or SCPC30 was significantlyhigher when these samples were seeded with cells than thatin the absence of cells. However, the Ca2þ concentrations inculture media were not significantly different for SCPC10and HA in the presence or absence of cells.

P concentration. Figure 1(c) shows the P ion concentrationof MEM (�) and MEM (þ) upon incubation with SCPC-, HA-,and TCPS-cell systems for 2 and 4 days. After 2 days, MEM(�) incubated with SCPC samples exhibited a significantincrease in the P ion concentration (41 ppm) compared tothe original MEM (�) solution in the order SCPC10 (51ppm) < SCPC 30 (62 ppm) < SCPC50 (77 ppm). On theother hand, MEM (�) incubated with HA had a P ion con-centration of 29 ppm, which was significantly lower thanthe original MEM (�) solution. The P ion concentration inoriginal MEM (þ) was about 225 % higher than that in theoriginal MEM (�) solution. The P ion concentration in MEM(þ) exhibited a significant increase after 2 days of incuba-tion with SCPC samples compared to the original MEM (þ)solution. Moreover, after 2 days, the P ion concentration inMEM (þ) incubated with SCPC50 (136 ppm) or SCPC30(128 ppm) was significantly higher than that in MEM (þ)incubated with SCPC10 (105 ppm), HA (112 ppm) or TCPS(86 ppm). MEM (þ) incubated with TCPS (86 ppm) showeda significant decrease in P ion concentration compared tothe original MEM (þ) (92 ppm). However, MEM (þ) incu-bated with HA exhibited P ion concentration comparable tothe original MEM (þ) solution after 2 days.

884 GUPTA, KIRAKODU, AND EL-GHANNAM EFFECTS OF EXOGENOUS PHOSPHORUS AND SILICON

After 4 days, P ion concentration in MEM (�) incubatedwith SCPC samples was comparable to that in the originalMEM (�) solution [Fig. 1(d)]. However, P ion concentrationin MEM (�) incubated with TCPS (33 ppm) or HA (27ppm) was significantly lower compared to the original MEM(�) solution. MEM (þ) incubated with all SCPC samplesshowed a significant increase in P-content compared to theoriginal MEM (þ) solution. Moreover, MEM (þ) incubatedwith SCPC50 exhibited significantly higher P ion concentra-tion than that incubated with any other sample after 4 days.

The P ion concentration in MEM (þ) incubated withSCPC50 (145 ppm) or SCPC30 (122 ppm) was significantlyhigher than that in MEM (þ) incubated with TCPS (106ppm) or HA (91 ppm).

ICP analysis also showed that P ion concentration in the cul-ture media [MEM (�) or MEM (þ)] after 2 and 4 days of incuba-tion with any of the SCPC samples was significantly higher inthe presence than in the absence of bone cells. In contrary, the Pion concentrations in the culture media incubated with HA pre-seeded with or without cells were not significantly different.

FIGURE 1. Ca2þ (a, b), phosphorus ion (c, d), and Si-ion (e, f) concentrations in tissue culture media MEM (þ) or MEM (–) after 2 and 4 days of

incubation with SCPC-, HA-, or TCPS-cell systems. MEM (þ) represents MEM supplemented with b-GP, while MEM (–) contained no exogenous

b-GP supplementation. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

ORIGINAL ARTICLE


Si concentration. Figure 1(e,f) represents the ICP analysisof Si released from SCPC after 2 and 4 days of incubation inMEM (�) or MEM (þ). Because the original media, HA andTCPS, do not contain any Si, there was no detectable Si forthese samples. After both 2 and 4 days of incubation inMEM (�) or MEM (þ), the Si released from SCPC increasedin the order SCPC10 < SCPC30 < SCPC50. Moreover, it wasalso observed that after 4 days of incubation in MEM (�),the Si-release from SCPC50 was significantly higher whenthe samples were seeded with cells compared to that in theabsence of cells.

Osteoblast gene expressionFigure 2 shows the ratios of target gene mRNA/b-actinmRNA for osteoblast-like cells attached to SCPC, HA, orTCPS after 2 days of incubation in media supplementedwith [MEM (þ)] or without [MEM (�)] 3 mM b-GP. Figure2(a) shows that after 2 days in culture, collagen-1/b-actinmRNA ratio on SCPC or TCPS incubated in MEM (�) orMEM (þ) was comparable. However, collagen-1/b-actinmRNA ratio on HA incubated in MEM (�) was significantlyhigher than that on HA incubated in MEM (þ). Moreover,OSN/b-actin mRNA ratios on SCPC10 or HA incubated in

MEM (�) were significantly higher than those on SCPC10 orHA incubated in MEM (þ) after 2 days in culture [Fig. 2(b)].In contrast, OPN/b-actin mRNA ratios on SCPC, HA, or TCPSwere significantly higher upon incubation in MEM (þ) thanthat in MEM (�) [Fig. 2(c)]. Moreover, among the samplesincubated in MEM (þ), cells attached to Si-rich SCPC50expressed significantly higher OPN/b-actin mRNA ratiosthan that on SCPC10, SCPC30, HA, or TCPS. In addition,OPN/b-actin mRNA ratio was significantly higher on SCPCsamples compared to that on TCPS for both MEM (þ) andMEM (�) culture conditions. Figure 2(d) shows after 2days, OCN/b-actin mRNA ratios on SCPC10, SCPC30, and HAwere significantly higher upon incubation in MEM (þ) thanthat in MEM (�). Moreover, the OCN/b-actin mRNA ratio onSCPC50 was significantly higher than that on other SCPC,HA, or TCPS samples incubated in MEM (�) or MEM (þ)culture conditions.

Figure 3 illustrates the gene expression of osteoblast-like cells on SCPC, HA, or TCPS after 4 days of incubation inMEM (þ) or MEM (�). The collagen-1/b-actin mRNA ratioson SCPC samples incubated in MEM (þ) were significantlylower compared to that on SCPC samples incubated in MEM(�) [Fig. 3(a)]. However, collagen-1/b-actin mRNA ratios onHA or TCPS upon incubation in MEM (þ) were comparable

FIGURE 2. Quantitative real-time PCR analysis of the levels of osteogenic mRNA for (a) Col-l, (b) OSN, (c) OPN, and (d) OCN expressed by neo-

natal rat calvaria osteoblast-like cells attached onto the surface of SCPC, HA, and TCPS after 2 days of incubation in MEM (–) or MEM (þ). The

mRNA expression level of OCN and OPN was significantly greater on Si-rich SCPC50 than that on other SCPC, HA, or TCPS samples cultured in

similar conditions. This suggested that osteoblast differentiation was preferentially enhanced on the Si-rich SCPC substrates. Moreover, OPN

and OCN expression on SCPC, HA, and TCPS was significantly higher in MEM (þ) compared to that in MEM (�). Although OCN expression on

SCPC50 in MEM (�) and MEM (þ) was comparable. These results indicated that up to 48 h, phosphate supplementation helped in advancing the

osteoblast lineage toward differentiation.


to that in MEM (�). Among the samples incubated in MEM(þ) for 4 days, the collagen-1/b-actin mRNA and OSN/b-actin mRNA ratios on TCPS were significantly higher thanthat on SCPC or HA [Fig. 2(b)]. Moreover, OSN/b-actinmRNA, OPN/b-actin mRNA, and OCN/b-actin mRNA ratioson SCPC samples incubated in MEM (þ) were significantlylower than that on SCPC samples incubated in MEM (�)[Fig. 3(b–d)]. However, for HA and TCPS, the OSN, OPN, orOCN mRNA expression in MEM (þ) and MEM (�) werecomparable, respectively. Among all the samples incubatedfor 4 days in MEM (�), the OPN/b-actin mRNA and OCN/b-actin mRNA ratios on Si-rich SCPC50 are significantly higherthan that on other SCPC, HA, or TCPS samples.

DISCUSSION

SCPC composites have a porous resorbable matrix and ex-hibit controlled dissolution in physiological solutions thatlead to enhanced osteoblast differentiation. This was evidentby the significant upregulated expression of OCN and OPNmRNA by osteoblast-like cells attached to Si-rich SCPC sam-ples compared to that expressed by cells attached to HA orTCPS upon incubation in MEM (�). It was observed that inabsence of exogenous phosphate supplementation, cells

attached to Si-rich SCPC rapidly advanced toward differen-tiation stage. In contrast, cells attached to Si-poor SCPC, HA,or TCPS samples primarily expressed markers related toproliferation stage. These observations suggested a possiblerole played by Si in stimulating bone-cell differentiation. Itwas also noted that when the media was exogenously sup-plemented with b-GP, the osteoblast phenotypic expressionon SCPC samples was significantly downregulated after 4days. This was attributed to the elevated phosphorus levelsin the culture media due to the additional dissolution of Pions from the SCPC. Such an effect was not observed forcells attached to HA and incubated with media supple-mented with b-GP due to the minimal dissolution of the HAin the culture media. Additionally, it was noted that the dis-solution of SCPC was enhanced in the presence of cells, indi-cating that the material dissolution was both a cell- and so-lution-mediated phenomenon.

The Ca2þ concentrations in culture media incubatedwith SCPC-cell systems were either similar to the originalmedia or showed minimal increase after incubation for 2and 4 days (Fig. 1). This indicated equilibrium between thedissolution and back precipitation of Ca2þ at the SCPC-solu-tion interface. ICP analysis also showed that the Ca2þ

FIGURE 3. (a) Col-l, (b) OSN, (c) OPN, and (d) OCN gene expression on SCPC, HA, and TCPS samples incubated in MEM (�) or MEM (þ) for 4

days. After 4 days of incubation in MEM (�), cells attached to SCPC50 exhibited significantly upregulated mRNA expression of OCN and OPN,

markers for bone-cell differentiation. On the other hand, cells on TCPS incubated in MEM (�) primarily expressed proliferation markers, Col-1

and OSN. The osteoblast gene expression on SCPC was significantly decreased when samples were incubated in MEM (þ) compared to that in

MEM (�). However, the gene expression on HA or TCPS was comparable in MEM (�) and MEM (þ). In general, the gene expression on SCPC

and HA samples was greater than that on TCPS, indicating that the 3D topography and chemical cues provided by the bioceramic substrate

enhanced bone-cell function.

ORIGINAL ARTICLE


concentration in media incubated with Si-rich SCPC50 wassignificantly higher in the presence of cells than that in theabsence of cells. This indicated that the cellular activity onSCPC50 enhanced the material dissolution over a 4-day pe-riod. In contrast, the 4-day incubation period and/or thecellular activity on HA were not sufficient to induce cell-mediated resorption of the material. Culture media incu-bated with HA showed a significant decrease in Ca2þ con-centrations after 2 and 4 days of incubation. In conjunctionwith the decrease in Ca ion concentration, media incubatedwith HA samples showed a decrease in P-content comparedto the original media. This can be attributed to the back pre-cipitation of P and Ca from the medium onto the surface ofHA. It is also interesting to note that due to the high-chemicalstability of HA, the attached cells did not contribute to anychanges in the P or Ca content of the media. On the otherhand, bone cells attached to SCPC enhanced the resorption ofthe material as indicated by the significant increase in P ionconcentrations in media after 2 and 4 days of incubation.The dissolution of NaCaPO4 of SCPC would result in therelease of Ca and P in a ratio 1:1, however, as the back pre-cipitation of HA (Ca/P ¼ 1.67) surface layer takes place itresults in less P uptake than Ca. It was also observed thatthe P ion concentration in media incubated with SCPC was inthe order SCPC10 < SCPC30 < SCPC50, even though the ini-tial P-content in SCPC10 is higher than that in SCPC30,which, in turn, is greater than that in SCPC50. This is inagreement with the results reported earlier26 and can beattributed to the differences in the phase composition andultrastructure that induce an increased rate of bioactivityreaction as the Si-content in SCPC is increased.14

The significantly higher release of Si ions from SCPC50compared to that from SCPC30 or SCPC10 after 2 and 4days of incubation in physiological solution (Fig. 1) is inagreement with earlier reported results26 and could beattributed to several factors. First, the initial Si-content ofSCPC50 is greater than the other two SCPC compositionstested. Thus, a higher concentration gradient would be adriving force for greater Si release from SCPC50. Second,SCPC50 has been shown to contain highest nanoporosityamong all the SCPC compositions tested,28 which signifi-cantly increases the surface area of SCPC50 in contact withthe physiological solution, thereby increasing the materialdissolution. Third, the crystalline phase composition of allthe SCPC compositions tested are different,14 and SCPC50contains NaCaPO4 phase, which is known to exhibit high-matrix reactivity in physiological solutions.14 Last, silicate–phosphate ion substitution at grain boundaries and triplejunctions in the material lead to enhanced material dissolu-tion.32 A combination of these factors may lead to increasedmatrix reactivity of Si-rich SCPC50 and enhance its dissolu-tion when contacted by physiological fluids. It was alsoobserved that the Si-release from SCPC50 incubated in MEM(�) for 2 and 4 days was significantly higher in the pres-ence of cells than that in the absence of cells, owing to thecell-mediated dissolution of the material.

The OCN/b-actin mRNA ratio on Si-rich SPC50 was six-fold higher than that on HA after 2 days of incubation in

MEM (�) (Fig. 2). On the other hand, Collagen-I and OSNmRNA expression was significantly higher on HA than thaton SCPC50 after 2 days of incubation in MEM (�). Thisdemonstrated that the cells attached to Si-rich SCPC50 wereproducing osteoblast differentiation markers while thoseattached to HA expressed markers for proliferation.33,34

Thus, there is a possible role played by the substrate aswell as the dissolved Si from SCPC50 that enhanced bone-cell differentiation. The expression of OPN and OCN mRNAon SCPC, TCPS, or HA samples incubated in MEM (þ) washigher than that in MEM (�) after 2 days (Fig. 2). This sug-gested that, initially, the b-GP supplementation in the cul-ture media had a stimulatory effect on bone-cell differentia-tion. In fact, it has been reported that under certainexperimental conditions the presence of phosphorus hasbeen shown to stimulate OPN mRNA expression.17,19 In gen-eral, it was observed that, after 4 days, the osteoblast geneexpression on SCPC samples was significantly lower whenthe samples were cultured in MEM (þ) compared to that inMEM (�) (Fig. 3). However, the gene expression on HA inMEM (þ) and MEM (�) was comparable after 4 days. More-over, the gene expression on TCPS in MEM (þ) or MEM (�)was also comparable after 4 days. It has been reported thatat elevated Pi levels, Ca

2þ-Pi ion pair can act as a potentosteoblast apoptogen.22 It was observed that at Pi concen-tration of 5 mM, an otherwise normal Ca2þ concentrationsof 2.8 mM, induced skeletal cell apoptosis after only 24 h.However, It was noted that if the Pi concentration was lowerthan 2 mM, then the osteoblasts remained vital even athigh-Ca2þ concentrations of 10 mM. In another study, it wasshown that when the osteoblasts were exposed to 5 mM Piconcentration for 4 days, the cell viability was decreased by70%.20 Furthermore, in an in vivo study, inhibition of boneregeneration was attributed to the local elevated Pi concen-tration resulting from dissolution of b-TCP.35 These studiesconcluded that sustained exposure of osteoblast to elevatedP ion concentration or to a combination of Ca and P ionconcentrations in certain ranges can lead to reduced cellfunction and may even cause cell death. In this study, b-GPsupplementation and the ionic dissolution from SCPC led asignificant increase in the P content of the media [MEM(þ)].Thus, a sustained exposure of osteoblast-like cellsattached to SCPC samples to elevated P levels in MEM (þ)could have adversely affected the viability of these cells,which, in turn, led to a significant downregulation of osteo-blast gene expression on SCPC after 4 days of incubation inMEM (þ). In contrast, there was no significant increase in Pion concentration in MEM (þ) incubated with HA or TCPS. Inaddition, MEM (þ) samples incubated with HA samples exhib-ited a significant decrease in Ca2þ concentrations after both 2and 4 days. Therefore, cells attached to HA and incubated inMEM (þ) are not exposed to as high-P levels as thoseattached to SCPC over the 4-day period. Moreover, the Ca2þ

concentration in MEM (þ) incubated with HA is also signifi-cantly lower than that incubated with SCPC samples. Thus,the gene expression of cells seeded on HA and incubated inMEM (þ) was not significantly affected after 4 days becauseof the combination of lower Ca and P ion concentrations.


As shown in Figure 1, the downregulation of osteoblastgene expression due to elevated ionic concentrations in themedia was not an issue in MEM (�). In such cases whereexternal factors like b-GP are absent, the stimulus for osteo-blast differentiation will primarily come from the materialsubstrate and its dissolution products. It was observed thatafter 4 days of incubation in MEM (�), the osteoblast phe-notypic expression was maximally upregulated on Si-richSCPC50 composite (Fig. 3). SCPC50 has been shown to becharacterized by a nanoscale ultrastructure and is rich insilica, which is a constitutive phase in the material ma-trix.14,29 It has been reported in literature that osteoblastfunction is significantly enhanced on nanophase materialscompared to that on their conventional counterparts.36

Moreover, previous work from our laboratory has demon-strated that osteoblast-like cells seeded on SCPC preferen-tially deposit extracellular matrix along the silica-richphases on the material surface.14 Thus, the unique combina-tion of chemistry and ultrastructure enabled Si-rich SCPC50to naturally enhance osteoblast function. After 4 days ofincubation in MEM (�), the ratios of OCN/b-actin mRNA andOPN/b-actin mRNA were significantly higher for cellsattached to Si-rich SCPC50 compared to that on other sam-ples. As OCN and OPN are markers for osteoblast differentia-tion,33,34 these results showed that Si-rich SCPC50 compositestrongly stimulated bone-cell differentiation. It also suggestsa role played by dissolved Si in enhancing the differentiationof osteoblast-like cells. These results are in corroborationwith earlier studies that have demonstrated the osteoinduc-tive potential of Si in enhancing bone-cell function.12,14,29,31

The gene expression on SCPC and HA samples was signifi-cantly greater than that on TCPS after 4 days of incubation inMEM (�). This could be attributed to the 3D topography andchemical cues provided by the bioceramic substrate and dis-solution products that enhance bone-cell function.

CONCLUSION

SCPC composites exhibited a controlled cell- and solution-mediated dissolution in cell-culture media over a 4-day pe-riod. In contrast, crystalline HA samples exhibited minimalcell- or solution-mediated dissolution over the same period.Cells attached to Si-rich SCPC composites most rapidlyadvanced to the differentiation stage as indicated by high-osteopontin and osteocalcin mRNA expression, suggesting arole played by Si in enhancing bone-cell differentiation.b-GP supplementation, in conjunction with ionic dissolutionfrom SCPC, led to significantly elevated P levels in themedia, which, in turn, resulted in downregulation of osteo-blast gene expression. In the absence of exogenous supple-ments, the chemical cues provided by SCPC dissolutionproducts were sufficient to stimulate bone-cell differentia-tion. Thus, a unique combination of composition and ultra-structure enabled SCPC to provide a natural stimulus toenhance bone-cell function in vitro.

ACKNOWLEDGMENTS

The authors thank Interpore Cross, Irvine, CA, for their gener-ous donation of ProOsteonTM hydroxyapatite discs.

REFERENCES1. Ducheyne P, Qui Q. Bioactive ceramics: The effect of surface reac-

tivity on bone formation and bone cell function. Biomaterials

1999;20:2287–2303.

2. de Bruijn JD, Bovell YP, van Blitterswijk CA. Structural arrange-

ments at the interface between plasma sprayed calcium phos-

phates and bone. Biomaterials 1994;15:543–550.

3. Alsberg E, Kong HJ, Hirano Y, Smith MK, Albeiruti A, Mooney DJ.

Regulating bone formation via controlled scaffold degradation.

J Dent Res 2003;82:903–908.

4. Hench LL. Bioceramics: From concept to clinic. J Am Ceram Soc

1991;74:1487–1510.

5. Ducheyne P, LeGeros RZ, Nery E, Lynch K, Kerebel B. Transfor-

mation of biphasic calcium phosphate ceramics in vivo: Ultra-

structural and physicochemical characterization. J Biomed Mater

Res 1989;23:883–894.

6. Ducheyne P, Radin S, King L. The effect of calcium-phosphate ce-

ramic composition and structure on in vitro behaviour. I. Dissolu-

tion. J Biomed Mater Res 1993;27:25–34.

7. Hing KA. Bioceramic bone graft substitutes: Influence of porosity

and chemistry. Int J Appl Ceramic Tech 2005;2:184–199.

8. Radin S, Ducheyne P, Berthold P, Decker S. Effect of serum pro-

teins and osteoblasts on the surface transformation of a calcium

phosphate coating: A physicochemical and ultrastructural study.

J Biomed Mater Res 1998;39:234–443.

9. Radin S, Ducheyne P. Effect of serum proteins on solution-

induced surface transformations of bioactive ceramics. J Biomed

Mater Res 1996;30:273–279.

10. de Groot K. Ceramics of calcium phosphates: Preparation and

properties. In: de Groot K, editor. Bioceramics of Calcium-Phos-

phate. Boca Raton, FL: CRC Press; 1983. p 99–114.

11. Eggli PS, Muller W, Schenk RK. Porous hydroxyapatite and trical-

cium phosphate cylinders with two different pore size ranges

implanted in the cancellous bone of rabbits. Clin Orthop 1988;

232:127–137.

12. Xynos ID, Edgar AJ, Buttery LD, Hench LL, Polak JM. Ionic prod-

ucts of bioactive glass dissolution increase proliferation of human

osteoblasts and induce insulin-like growth factor II mRNA expres-

sion and protein synthesis. Biochem Biophys Res Commun 2000;

276:461–465.

13. Reffitt DM, Ogston N, Jugdaosingh R, Cheung HFJ, Evans BAJ,

Thompson RPH, Powel JJ, Hampson GN. Orthosilicic acid stimu-

lates collagen type 1 synthesis and osteoblastic differentiation in

human osteoblast-like cells in vitro. Bone 2003;32:127–135.

14. El-Ghannam A, Ning CQ. Effect of bioactive ceramic dissolution

on the mechanism of bone mineralization and guided tissue

growth in vitro. J Biomed Mater Res 2006;76:386–397.

15. Yamaguchi T, Chattopadhyay N, Kifor O, Butters RR, Sugimoto T,

Brown EM. Mouse osteoblastic cell line (MC3T3-E1) expresses

extracellular calcium (Ca2þ)-sensing receptor and its agonists

stimulate chemotaxis and proliferation of MC3T3-E1 cells. J Bone

Miner Res 1998;13:1530–1538.

16. Ma A, Yang Y, Carnes DL, Kim K, Park S, Oh SH, Ong JL. Effects

of dissolved calcium and phosphorus on osteoblast responses.

J Oral Implantol 2005;31:61–67.

17. Beck GR Jr., Zerler B, Moran E. Phosphate is a specific signal for

induction of osteopontin gene expression. Proc Natl Acad Sci

USA 2000;97:8352–8357.

18. Beck GR, Moran E, Knecht N. Inorganic phosphate regulates mul-

tiple genes during osteoblast differentiation, including Nrf2. Exp

Cell Res 2003;288:288–300.

19. Beck GR. Inorganic phosphate as a signaling molecule in osteo-

blast differentiation. J Cell Biochem 2003;90:234–243.

20. Meleti Z, Shapiro IM, Adams CS. Inorganic phosphate induces ap-

optosis of osteoblast-like cells in culture. Bone 2000;27:359–366.

21. Adams CS, Shapiro IM. Mechanisms by which extracellular matrix

components induce osteoblast apoptosis. Connect Tissue Res

2003;44(Suppl 1):230–239.

22. Adams CS, Mansfield K, Perlot RL, Shapiro IM. Matrix regulation

of skeletal cell apoptosis. Role of calcium and phosphate ions.

J Biol Chem 2001;276:20316–20322.

23. Bellowa CG, Heersche JNM, Aubin JE. Inorganic phosphate

added exogenously or released from b-glycerphosphate initiates

ORIGINAL ARTICLE


mineralization of osteoid nodules in vitro. Bone Miner 1992;17:

5–29.

24. Khouja HI, Bevington A, Kemp GJ, Russel RGG. Calcium and

orthophosphate deposits in vitro do not imply osteoblast-medi-

ated mineralization: Mineralization by betaglycerophosphate in

absence of osteoblasts. Bone 1990;11:385–391.

25. Boskey AL, Guidon P, Doty SB, Stiner D, Leboy P, Binderman I.

The mechanism of b-glycerophosphate action in mineralizing

chick limb-bud mesenchymal cell cultures. J Bone Miner Res

1996;11:1694–1702.

26. Gupta G, Kirakodu S, El-Ghannam A. Dissolution kinetics of a Si-

rich nanocomposite and its effect on osteoblast gene expression.

J Biomed Mater Res A 2007;80:486–496.

27. El-Ghannam A, Ning C, Mehta J. Cyclosilicate nanocomposite: A

novel resorbable scaffold for BMP and bone marrow cell delivery.

J Biomed Mater Res A 2004;71:377–390.

28. Gupta G, El-Ghannam A, Kirakodu S, Khraisheh M, Zbib H.

Enhancement of osteoblast gene expression by mechanically

compatible porous Si-rich nanocomposite. J Biomed Mater Res B

Appl Biomater 2007;81:387–396.

29. Ning CQ, Mehta J, El-Ghannam A. Effects of silica on the bioactiv-

ity of calcium phosphate composites in vitro. J Mater Sci Mater

Med 2005;16:355–360.

30. El-Ghannam A, Ducheyne P, Shapiro IM. Formation of surface

reaction products on bioactive glass and their effects on the

expression of the osteoblastic phenotype and the deposition of

mineralized extracellular matrix. Biomaterials 1997;18:295–303.

31. Knabe C, Stiller M, Berger G, Reif D, Gildenhaar R, Howlett CR,

Zreiqat H. The effect of bioactive glass ceramics on the expres-

sion of bone-related genes and proteins in vitro. Clin Oral Implant

Res 2005;16:119–127.

32. Porter AE, Patel N, Skepperb JN, Besta SM, Bonfield W. Compari-

son of in vivo dissolution processes in hydroxyapatite and sili-

con-substituted hydroxyapatite bioceramics. Biomaterials 2003;24:

4609–4620.

33. Rodan GA, Noda M. Gene expression in osteoblastic cells. Crit

Rev Eukaryot Gene Expr 1991;1:85–98.

34. Aubin JE. Advances in osteoblast lineage. Biochem Cell Biol

1998;76:899–910.

35. Eid K, Zelicof S, Perona BP, Sledge CB, Glowacki J. Tissue reac-

tions to particles of bone-substitute mateials in intraosseous and

heterotopic sites in rats: Discrimination of osteoinduction, osteo-

compatibility and inflammation. J Orthop Res 2001;19:962–969.

36. Webster TJ, Ergun C, Doremus RH, Siegel RW, Bizios R.

Enhanced functions of osteoblasts on nanophase ceramics. Bio-

materials 2000;21:1803–1810.


Effects of exogenous phosphorus and silicon on osteoblast differentiation at the interface with...

Documents

Transcript of Effects of exogenous phosphorus and silicon on osteoblast differentiation at the interface with...