Potential pitfalls of radiolabel adsorption to ceramicbiomaterials

Tony Parker,1 Zee Upton,1 Douwe Vellinga,1,* Mei Wei,2,† David Leavesley1

1Tissue BioRegeneration Domain, Institute of Health and Biomedical Innovation and the School of Life Sciences,Queensland University of Technology, GPO Box 2434, Brisbane, Queensland 4001, Australia2The School of Mechanical, Manufacturing and Medical Engineering, Queensland University of Technology, Brisbane,Queensland 4001, Australia

Received 23 August 2004; accepted 8 September 2004Published online 24 January 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.30247

Abstract: The use of radiolabeled precursor molecules forthe metabolic analysis of cell functions is commonplace.Tritiated thymidine, in particular, has been used to quanti-tate cellular proliferation in numerous cells, including osteo-blasts, when cultured on various biomaterials. Our aim wasto assess cellular protein synthesis and proliferation, on arange of fluoride ion-substituted hydroxyapatites. Initially,we used a classical metabolic analysis strategy with radio-labeled tracer molecules. Our results suggested that thesematerials supported enhanced protein synthesis and prolif-eration of SaOS-2 human osteoblast-like cells. However,control samples also revealed enhanced adsorption of theradiolabeled tracer. We have shown that this arises becausepartially fluoride ion-substituted hydroxyapatite exhibitsenhanced adsorptive characteristics of radiolabeled leucine

and thymidine over tissue culture plastic, hydroxyapatite,and fluoroapatite. Moreover, manual cell count data ob-tained through SEM analysis showed no significant differ-ence in cell proliferation between any of the materials, fur-ther indicating that our initial results were artifacts. Theseresults highlight the use and reporting of appropriate cell-free controls are critical in bioassays examining functionalresponses of cells to biomaterials, and if absent, may con-found accurate data interpretation. Our findings have gen-eral implications for investigations of cell function on othernovel ceramic biomaterials. © 2005 Wiley Periodicals, Inc.J Biomed Mater Res 72A: 363–372, 2005

Key words: adsorption; biomaterial; ceramic; osteoblast;proliferation

INTRODUCTION

Metabolic labeling of DNA or protein in culturedcells with radioactive, low molecular weight precur-sors is a popular technique used widely for the in vitroanalysis of macromolecule biosynthesis. In particular,the detection of incorporated tritiated thymidine([3H]-thy) as a measure of DNA synthesis, and thusproliferation of cultured cells, has been used for manyyears.1,2 Moreover, cellular incorporation of selectedradiolabeled amino acids (e.g., leucine or methionine)into newly synthesised protein has been shown tocorrelate with an increase in protein synthesis and/or

cell number.3–5 Indeed, tritiated thymidine uptake andincorporation is commonly used in the field of tissueengineering for quantitation of cellular proliferationon biomaterial scaffolds or substrates.6–9 Similarly,cellular protein synthesis has been quantitated by us-ing radiolabeled amino acids in cultures grown onbiomaterial substrates.9–13

Calcium phosphate ceramics have been investigatedfor many years as potential bone substitute materialsfor orthopedic applications, including the following:coatings for metal joint prostheses14,15; space-fillingpacking morsels; bone cements for use in reconstruc-tive surgery of bone defects arising from disease ortrauma16,17; and in other biomedical applications suchas antibiotic and anticancer drug delivery sys-tems.18–22 The latter is due to the excellent “bioaffin-ity” shown by calcium phosphate materials, and thishas also influenced their selection as adsorbents forliquid chromatography applications.23–25

Cellular interactions with the ceramic surface dependon a range of physical and chemical factors, includingthe surface topography and surface energy, which influ-ence the adsorptive capacity of the material for pro-

*Present address: Gausstraat 9, 6533, LD, Nijmegen, TheNetherlands

†Present address: Department of Metallurgy and MaterialsEngineering, Institute of Materials Science, 97 North Ea-gleville Rd., Unit 3136, University of Connecticut, Storrs, CT06269-3136, USA

Correspondence to: Tony Parker; e-mail: a0.parker@student.qut.edu.au

© 2005 Wiley Periodicals, Inc.

tein.6,26–29 Hydroxyapatite (Ca10(PO4)6(OH)2)(HA) is acalcium phosphate ceramic, chemically identical to themineral component of natural bone, and is used widelyas a bone substitute biomaterial in clinical applications.However, degradation and dissolution of HA implantcoatings observed in vivo30 have prompted considerableresearch to seek to improve the stability and bioactiveproperties of HA and similar ceramic bone substitutes.This research has included in recent times the incorpo-ration of fluoride ions into various materials includingHA.26,31–35 Several studies have shown that partial sub-stitution of the hydroxyl groups of hydroxyapatite withfluoride confers improved strength and durability to thematerial in vivo.34

One of the original aims of the present study was toassess the biocompatibility of a range of fluoride ion-substituted hydroxyapatites. This assessment initiallyincluded evaluation of de novo protein synthesis mea-sured by tritiated leucine ([3H]-leu) incorporation intonew protein(s) by the human osteoblast-like cell line,SaOS-2, concluding that an observed increase in pro-tein synthesis on the fluoride ion-substituted hydroxy-apatites could have been due to an increase in cellproliferation.36 However, subsequent studies as re-ported here show that [3H]-leu and [3H]-thy, bothwidely used for the metabolic labeling of culturedcells grown on biomaterial substrates, can adsorb non-specifically to the biomaterial, potentially leading toinaccurate interpretations of cellular protein synthesisand proliferation responses to the substrate. We alsoshow that the partially fluoride ion-substituted HAexhibits enhanced absorptive characteristics over HAand fluoroapatite (FA).

MATERIALS AND METHODS

Cells and culture conditions

Human osteoblast-like SaOS-2 cells, originally isolatedfrom a human osteosarcoma, were obtained from the Amer-ican Type Culture Collection (Rockville, MD; HTB-85). Thecells were maintained in minimal essential medium, alphaformulation (�-MEM) (Gibco, Auckland, NZ), supple-mented with 10% fetal calf serum (Gibco) 50 U/mL penicil-lin/streptomycin (Gibco), and 5 �g/mL gentamycin(Gibco). Cultures were discarded after 20 passages to ensurethe phenotype of the cell population was maintained.

Biomaterial disks

The biomaterials examined in this study were manufacturedand provided by the School of Mechanical, Manufacturing andMedical Engineering, Queensland University of Technology(QUT), as described elsewhere.36 Briefly, these were comprised

of hydroxyapatite (HA) (nominally Ca10(PO4)6OH2), fluoro-apatite (FA) (nominally Ca10(PO4)6F2) and three fluoride ion-substituted apatites: low [F�], medium [F�], and high [F�].Each biomaterial was compressed into disks for biological anal-yses, �0.7 cm2 by �2 mm. Assays of protein synthesis, de-scribed below, were performed by using newly manufactureddisks. These disks were then cleaned before reuse in the ad-sorption assays.

Previous experiments in our laboratory established thateffective cleaning of cellular debris from the surface of thevarious biomaterials can be achieved by rinsing the disksliberally with double distilled water (ddH2O) after 0.5MNaOH, 0.1% Triton X100 (Sigma, St Louis, MO) treatmentwithin each assay. The disks were then ultrasonicallycleaned with 3 � 10 min cycles in sterile distilled water, withsterile water exchanged between cleaning treatments. Diskswere blot-dried and the surface areas were calculated byusing a Hewlett Packard Scanjet 5470c scanner (HewlettPackard, Palo Alto, CA) and image analysis software (ScionImage, Frederick, MD) before dry heat sterilization at�160°C for 2 h before reuse.

Cellular protein synthesis assays

Protein synthesis by SaOS-2 cells was measured in tripli-cate with cultures grown for 24 h on each material type bydetermining [4,5-3H]-leucine ([3H]-leu) (Amersham, Buck-inghamshire, UK) incorporation into newly synthesized pro-tein. Experiments in our laboratory have confirmed that thisassay correlates well with increases in cell number (data notshown). New material disks of HA, low [F�], medium [F�],high [F�] and FA were placed into a 24-well polystyrenetissue culture plate (Nalge Nunc, Rochester, NY). One mil-liliter of growth media containing 2.5 � 105 or 1 � 105

SaOS-2 cells and 1 �Ci [3H]-leu (Amersham) was placed intothe wells and incubated in a humidified 5% CO2/95% airatmosphere at 37°C. Cells seeded into wells without disks(PL), as well as disks exposed to radiolabel but without cells,were used as controls. After 24 h, media containing nonad-herent cells and unincorporated radiolabel were removed,and the disks were transferred into a fresh 24-well plate.Cellular protein on the disks was precipitated with twowashes of 5% (w/v) trichloroacetic acid (BDH, Poole, UK)and subsequently solubilized with 1 mL of 0.5M NaOH,0.1% Triton X100 (Sigma) per well. Plates were then placedon an orbital shaker for at least 1 h, and triplicate 100-�Lsubsamples were transferred into individual vials contain-ing 5 mL of “Readysafe” scintillation cocktail (Beckman,Inc., Fullerton, CA) Radioactivity was determined in a Beck-man LS5000TA liquid scintillation counter (Beckman). Theresulting data were corrected by using data from the cell-free control samples and expressed as percentage of theeffect obtained with cells cultured on the polystyrene control(PL).

[3H]-leucine and [3H]-thymidine adsorption

Small molecule adsorption by each biomaterial was as-sessed from three disks of each material type in duplicate

364 PARKER ET AL.

assays. Briefly, 1 mL of phosphate-buffered saline (PBS;Oxoid, Hampshire, UK) containing 1 �Ci/mL [3H]-leu or[3H]-thy (Amersham) was transferred into each well of 24-well polystyrene plates (Nalge Nunc), which had been fittedwith a single HA, low [F�], medium [F�], high [F�] or FAdisk/well and incubated in a humidified 5% CO2/95% airatmosphere at 37°C. PBS containing radiolabel, transferredinto wells without disks, as well as disks incubated withradiolabel-free PBS were used as controls. After 24 h, PBSand excess radiolabel were removed, the disks were trans-ferred to a fresh 24-well plate, and then washed twice withradiolabel-free PBS. Adsorbed radiolabel was liberated witha 1-h treatment of 1 mL 0.5M NaOH, 0.1% Triton X100(Sigma) per well. Determination of adsorbed radiolabel wasconducted by scintillation counting as detailed above inCellular Protein Synthesis Assays.

SEM analysis of cell proliferation and morphology

For SEM analysis of SaOS-2 cell proliferation, duplicate,cleaned, sterile HA, low [F�], medium [F�], high [F�] andFA disks were placed into individual wells of a 24-wellpolystyrene plate. One milliliter of SaOS-2 cells in growthmedia was seeded onto each disk at a density of 1 � 105

cells/well and incubated in a humidified 5% CO2/95% airatmosphere at 37°C for 24 h. Cells seeded onto Thermanox™coverslips (PL) (Nalge Nunc) were used to compare cellproliferation and morphology under control conditions withthose on the various ceramic biomaterials. After a 24-h in-cubation, the growth medium was removed, and the diskswere transferred into a fresh 24-well plate and washed twicewith fresh PBS. The attached cells were fixed with 3% v/vglutaraldehyde in 0.1M sodium cacodylate buffer solution(pH 7.3) for 1 h at 4°C. Fixed specimens were then dehy-drated in serial alcohols; two changes each of 50%, 70%, 90%,and 100% ethanol were incubated for 10 min between eachchange. Specimens were then critical point dried (DentonVacuum, Moorestown, NJ) and gold coated in a SC500,Bio-Rad sputter coater (Bio-Rad, Hercules, CA) before ex-amination using an FEI Quanta 200 scanning electron mi-croscope (FEI, Hillsboro, OR). Images of at least eight ran-dom fields of cells per disk were captured at either �400,�800, or �1600 magnification, and all visible cells in eachimage were counted. The surface area of each image wascalculated, and the cell number was expressed as the num-ber of cells per cm2. Images collected at �2000 magnificationwere used to assess qualitative differences in cell morphol-ogy between each material.

Statistical analysis

Statistical analysis of adsorption assay data was per-formed by using Student’s t test to compare results obtainedfor each individual substrate type between duplicate assays.Subsequently, Tukey’s analysis was used to compare resultsobtained with the various substrates within each assay andfor SEM proliferation assay data.

RESULTS

Cellular protein synthesis

Protein synthesis is an integral element of all heal-ing processes, including bone remodeling. A success-ful biomaterial for skeletal implantation must there-fore be able to support osteoblast protein synthesis or“bioactivity.” The ability of each biomaterial to sup-port cellular “bioactivity” was assessed indirectlyfrom de novo protein synthesis as determined fromradiolabeled leucine incorporation into protein by at-tached cells. The partially fluorinated calcium phos-phates appeared to exhibit enhanced protein synthesiscompared with the control materials, tissue cultureplastic (polystyrene) (PL), and hydroxyapatite (HA)when cells were seeded at a density of 2.5 � 105

cells/well [Fig. 1(a)]. Specifically, defining the PL re-sults as 100%, the mean count per cm2 on HA was 7%below the PL control, low [F�] was 18% above thecontrol, medium [F�] was 33% above the control, high[F�] was 17% above the control, and FA was 6% belowthe PL control. However, when these results werecorrected with control data obtained from the cell-freecontrols (i.e., disks exposed to the radiolabel but withno cells present), the apparent enhanced protein syn-thesis on the partially fluorinated calcium phosphatesrelative to the PL control was abrogated [Fig. 1(b)].

Analysis of these data raised concerns that perhapscells cultured on the partially fluorinated biomaterialshad become confluent. This may cause contact inhibi-tion between cells within these cultures, potentiallyaltering cellular growth dynamics, leading to variantculture conditions between treatments over the courseof the assay. Hence, to test the effect of cell seedingdensity on protein synthesis potential, the experi-ments were repeated by using an initial cell seedingdensity of 1 � 105 cells/well. It is surprising that at thelower cell seeding density (1 � 105 cells/well), therelative protein synthesis of cells on the HA substratewas observed to be reduced compared with othersubstrates (44% of the PL control) [Fig. 2(a)]. Never-theless, the partially fluorinated materials did appearto support enhanced protein synthesis compared withHA and FA at this reduced seeding density. Specifi-cally, defining the PL results as 100%, the mean countper cm2 on low [F�] was 27% above the control, me-dium [F�] was 62% above the control, high [F�] was54% above the control, and FA was 30% below the PLcontrol. Again, however, when the results were cor-rected with control data obtained from the cell-freecontrols, the apparent enhanced protein synthesis ef-fect, relative to the PL control, was abrogated [Fig.2(b)]. Indeed, these corrected data indicate that therewas no enhanced protein synthesis on the partiallyfluorinated biomaterials over the PL control. More

RADIOLABEL ADSORPTION TO BIOMATERIALS 365

importantly, it seemed that a greater amount of [3H]-leu was adsorbed to the partially fluorinated bioma-terials than to either the tissue culture plastic, HA orFA. However, reducing the cell seeding density didresult in increases in the relative difference in proteinsynthesis of SaOS-2 cells to the ceramic materials,indicating that possible contact inhibition of cellgrowth at the higher seeding density may have oc-curred, as proposed above.

To ascertain if the results obtained from the [3H]-leuassays were leucine specific, a single [3H]-thy-basedassay was performed under identical conditions. Theresults obtained from the [3H]-thy assay corroboratedthe results obtained by using [3H]-leu and confirmedour observation of a large enhancement of cellular“activity” on the fluorinated biomaterials, comparedwith either tissue culture plastic or HA [Fig 3(a)].

However, on correcting these data with the cell-freecontrol data for these disks (disks exposed to radiola-bel in the absence of cells), the medium [F�] and high[F�] materials revealed large negative responses(�323% and �387% of the PL control, respectively)[Fig. 3(b)]. These data indicate that the partially fluor-inated hydroxyapatites adsorb and retain more [3H]-thy than either PL, HA, or FA and that the “apparent”enhanced proliferative effect is abolished when cor-rected with data obtained from the cell-free controls.

[3H]-leucine and [3H]-thymidine adsorption

When performing the above assays, we became con-cerned that [3H]-leu and [3H]-thy were nonspecifically

Figure 2. De novo protein synthesis of SaOS-2 cells onvarious biomaterials. De novo protein synthesis, as deter-mined by radiolabeled leucine incorporation into newly syn-thesised protein, was examined in SaOS-2 cells grown oneach biomaterial for 24 h after initial seeding density of 1.0 �105 cells/well. Assays were performed in duplicate withtriplicate cultures for each disk type. Results are expressedas a percentage of the tissue culture plastic control � SEMand depict (a) uncorrected data and (b) results correctedwith cell-free control data (i.e., test materials exposed toradiolabel, but in the absence of cells).

Figure 1. De novo protein synthesis of SaOS-2 cells onvarious biomaterials. De novo protein synthesis, as deter-mined by radiolabeled leucine incorporation into newly syn-thesised protein, was examined in SaOS-2 cells grown oneach biomaterial for 24 h after initial seeding density of 2.5 �105 cells/well. Assays were performed in duplicate withtriplicate cultures for each disk type. Results are expressedas a percentage of the tissue culture plastic control � SEMand depict (a) uncorrected data and (b) results correctedwith cell-free control data (i.e., test materials exposed toradiolabel, but in the absence of cells).

366 PARKER ET AL.

adsorbing to the partially fluorinated biomaterials,producing misleading results and confounding accu-rate interpretation. To test this hypothesis, we per-formed a series of cell-free adsorption assays using[3H]-leu and [3H]-thy. Figure 4(a,b) depicts the resultsfrom two separate assays in which triplicate disks ofeach material were tested for [3H]-leu adsorption.These results indicate that the counts adsorbed variedsignificantly between each individual assay as deter-mined by Student’s t test. Nevertheless, Tukey’s anal-ysis indicated that significantly more (p � 0.01) [3H]-leu adsorbed to medium [F�] in experiment A and Band low [F�] in experiment B, compared with any ofthe other materials tested. Figure 4(c,d) depicts theresults from two separate assays in which triplicatedisks of each material were tested for [3H]-thy adsorp-tion. These results indicate that the counts adsorbed

varied significantly between each individual assay asdetermined by Student’s t test. However, Tukey’sanalysis indicated that significantly more (p � 0.01)[3H]-thy adsorbed to medium [F�] in experiment Cand low [F�] in experiment D, compared with any ofthe other materials tested. The radiolabel-free controlswere within background radioactivity levels (data notshown).

Taken together, we conclude that the low [F�] andmedium [F�] substrates exhibit enhanced adsorptionof small biological molecules such as amino acidsand/or nucleotides relative to PL, HA, and FA.

SEM analysis of cell proliferation and morphology

Although fluorescent cell-labeling techniques maybe useful for quantitation of cell numbers on bioma-terials that are unable to be visualised with transmit-ted light, preliminary experiments revealed that theceramic disks exhibited varying auto fluorescence cor-responding to an increase in [F�], thus abrogating theapplication of fluorescence techniques to determinecell number in this study. Consequently, SEM analysiswas performed to confirm that there was in fact noenhanced proliferative effect when SaOS-2 cells weregrown on the partially fluorinated biomaterials. It isintriguing that the ceramic materials did appear tohave a slightly higher mean cell number per area thanthe plastic control (Fig. 5). Specifically, defining the PLresults as 100%, the mean cell density on HA was 32%above the PL control, low [F�] was 24% above thecontrol, medium [F�] was 27% above the control, high[F�] was 38% above the control, and FA was 25%above the PL control. However, Tukey’s analysis ofthese data determined that there was no significantdifference in cell number and density between any ofthe materials tested (Fig. 5). These data indicate thatthere was no enhanced proliferative effect on SaOS-2cells cultured on the partially fluorinated biomaterialsafter 24 h of culture. It is interesting that the morphol-ogy of cells grown on PL and HA were generally flatand well spread, whereas cells grown on low [F�] andhigh [F�] exhibited more rounded morphologies withmany fine filopodia (Fig. 6). Cells grown on the me-dium [F�] and FA substrates were heterogeneous;some cells appeared flat and well spread, whereasothers were more raised and rounded (Fig. 6). Thesedata suggest that the cells react differently to eachbiomaterial surface.

DISCUSSION

It is well known that the physical and chemicalcharacteristics of a biomaterial matrix or substrate sig-

Figure 3. Proliferation of SaOS-2 cells on various biomate-rials. Proliferation, as determined by radiolabeled thymidineincorporation into newly synthesised DNA, was examinedin SaOS-2 cells grown on each biomaterial for 24 h afterinitial seeding density of 1.0 � 105 cells/well. Assays wereperformed in duplicate with triplicate cultures for each disktype. Results are expressed as a percentage of the tissueculture plastic control � SEM and depict (a) uncorrecteddata and (b) results corrected with cell-free control data (i.e.,test materials exposed to radiolabel, but in the absence ofcells).

RADIOLABEL ADSORPTION TO BIOMATERIALS 367

nificantly influence the behavior of osteoblasts in cul-ture.6,26,29,37–46 Thus, the initial objective of this studywas to assess the biocompatibility of a range of fluo-ride ion-substituted hydroxyapatites, including exam-ining differences in cell protein synthesis and prolif-eration in response to the various materials. Becausethe SaOS-2 cell line has been shown to be “osteoblast-like”47 and has been widely used as a model for in-vestigation of osteoblast function,6,42,48 we used thesecells, as well as [3H]-leu as a molecular tag, to assaycellular protein synthesis. Leucine is an essentialamino acid and is, therefore, not produced endog-enously in humans and is required by human cellsalmost exclusively for use in protein synthesis.Leucine is also known to be metabolized to acetyl-CoA for oxidation in the citric acid cycle during star-vation or rigorous exercise.49 Therefore, [3H]-leu wasconsidered to be a suitable label for assessment ofcellular protein synthesis in the SaOS-2 cell line giventhat cells were cultured under nutrient rich conditions.

Actively growing cells were expected to take up the

[3H]-leu molecules from the medium and incorporatethem into newly synthesised protein during cellgrowth and division. However, the inclusion of cell-free controls in the assay protocol revealed unexpect-edly high radiotracer “activity” associated with thecell-free, partially fluorinated hydroxyapatites, indi-cating nonspecific adsorption of the radiolabel tothose substrates (Figs. 1 and 2). To determine if theobserved radiolabel binding was leucine specific, weperformed a single assay using [3H]-thy as the label.The large negative values obtained for the correcteddata supported the results of the [3H]-leu assays (Fig.3).

In a subsequent series of cell-free, adsorption assayswith either [3H]-leu or [3H]-thy (Fig. 4), we haveshown that both of these low molecular weight com-pounds, commonly used for the metabolic labeling ofcultured cells grown on biomaterial substrates, mayadsorb nonspecifically to the biomaterial, thus con-founding accurate analysis of the response of cells tosubstrates unless appropriate controls are used. More-

Figure 4. [3H]-leucine and [3H]-thymidine adsorption to various biomaterials. Radiolabel tracer adsorption to each bioma-terial was examined by incubation of individual biomaterial disks for 24 h with 1 mL PBS (pH 7.4) containing 1 �Ci/mL of[3H]-leucine (a) and (b) or [3H]-thymidine (c) and (d). Data are from two representative assays per isotope and expressed ascounts per minute/cm2 � SEM. Statistical significance (� p � 0.01) was determined by using Tukey’s test. The data havenot been pooled because of statistical differences between assays for some material types as determined by Students t test.

368 PARKER ET AL.

over, the actual protein or proliferative response ofSaOS-2 cells on each material could not be elucidatedby using this technique. In addition, it remains unclearwhether the attached cells were able to transport anduse the adsorbed [3H]-leu or [3H]-thy for protein orDNA synthesis. Peterson et al.50 recently reported thatpreosteoblast proliferation appeared to be inhibited incultures grown on extracellular matrix, in particulartype 1 collagen, but not on tissue culture plastic. Care-ful analysis of [3H]-thy incorporated into DNA andcell count data revealed no such inhibition; instead,they showed that thymidine transport and uptake hadbeen affected.50 Earlier, Maurer51 reviewed potentialproblems associated with the correlation between thy-midine uptake and cell proliferation, including theconversion of thymidine to metabolites other thannewly synthesised DNA and the possible interferencein the thymidine phosphorylation process by alkalinephosphatase. Well-known functions of osteoblasts in-clude the production of large amounts of both type 1collagen and alkaline phosphatase; thus, measurementof DNA synthesis using [3H]-thy appears to be espe-cially inappropriate for this cell type.

The mechanism for the adsorption of small mole-cules by ceramic biomaterials is not clear. However,Ohta et al.25 investigated the adsorption characteris-tics of -tricalcium phosphate, brushite and monetite,demonstrating that there are two classes of adsorbingsites on calcium phosphate particles, namely, posi-tively charged Ca2� sites that adsorb acidic proteinsand negatively charged PO4

� sites that adsorb basic

proteins. These data corroborate the earlier work ofKawasaki et al.23 who demonstrated that Ca2� andPO4

� sites exist on HA and were responsible for theadsorption of acidic and basic proteins, respectively.The results of these studies support our conclusionthat the adsorption of radiolabels to the partially flu-orinated biomaterials as found in the present studymay occur by a similar mechanism. Indeed, it is theseadsorptive properties of calcium phosphates, and par-ticularly HA, that have led to the use of these materi-als as solid-phase matrices for liquid chromatographyapplications.

Manual cell counts, obtained from SEM analysis ofabsolute adherent cell numbers on each material, re-vealed that cell proliferation was not significantly dif-ferent on any of the materials assessed. This observa-tion supports our interpretation that despite the initial“apparent” enhanced proliferative effect of the par-tially fluorinated hydroxyapatites, there is actually noenhanced proliferation of SaOS-2 cells on any of thebiomaterials over that found for PL (Fig. 5). However,differences in morphology of cells grown on PL andHA, compared with those grown on the fluorinatedsubstrates, indicate that the cells react differently to 1)variations in chemical characteristics between the sub-strates, 2) different surface textures, or 3) both (Fig. 6).The surface roughness of biomaterial matrices iswidely known to influence the differentiation pro-cesses of osteoblasts.6,26,37,42 Therefore, we suggestthat the partially fluorinated biomaterials tested heremay alter the differentiation processes of osteoblastscompared with that of PL or HA, although this hy-pothesis remains to be tested. In addition, these dataalso suggest that although the partially fluorinatedhydroxyapatites do not support enhanced proteinsynthesis or proliferation of osteoblasts, they mayhave potential as drug delivery vehicles for antibiot-ics, anticancer therapeutics, or growth factors in anorthopedic setting.

This conclusion is supported by studies involvingthe use of antibiotic-impregnated HA for the success-ful treatment of osteomyelitis in rats52 and humans.21

In addition, growth factors such as transforminggrowth factor-1 (TGF-1) and bone morphogeneticproteins (BMPs) have been adsorbed to calcium phos-phate ceramic implants and investigated for their ef-fects on bone formation in vivo. Indeed, Lind et al.53

found TGF-1/calcium phosphate composite im-plants stimulated both osteoclast and osteoblast activ-ity resulting in increased bone resorption and acceler-ated formation of woven bone around the implant.Similarly, Noshi et al.54 described enhanced osteogen-esis around porous HA implants that had been coatedwith BMPs and soaked in cultures of marrow cells,compared with either HA implants alone, HA im-plants and marrow cells alone, or HA implants and

Figure 5. SEM analysis of SaOS-2 cell proliferation. SaOS-2cells (1 � 105 cells/well per 24-well plate) were allowed toattach and proliferate on new biomaterial disks for 24 h . Thecultures were then washed twice with PBS, fixed with 3%glutaraldehyde, dehydrated, dried, and coated with gold ina Bio-Rad sputter coater before imaging. Random fieldswere imaged and all visible cells in each field were counted.The area of each image was calculated, and the cell numberwas expressed as number of cells per cm2. Results are ex-pressed as the mean number of cells per cm2 as a percentageof the plastic control � SEM. Tukey’s analysis determinedthere is no significant difference between each material type.

RADIOLABEL ADSORPTION TO BIOMATERIALS 369

BMPs alone before subcutaneous implantation intothe backs of rats.

CONCLUSION

In this study, we demonstrate that partially fluori-nated biomaterials, and in particular low [F�] and

medium [F�], show enhanced adsorption of [3H]-leuand [3H]-thy relative to PL, HA, or FA. Consequently,and most importantly, this study has raised concernswith the currently accepted practice of using lowmolecular weight biological tracer molecules for themetabolic analysis of cell functions, including proteinsynthesis and/or cell proliferation, on ceramic bioma-terials. Our findings clearly illustrate the need for

Figure 6. Morphology of SaOS-2 cells on various biomaterials. SaOS-2 cells (1 � 105 cells/well) were allowed to attach andproliferate on each substrate (PL (a), HA (b), Low [F�] (c), Medium [F�] (d), High [F�] (e) or FA (f)) for 24 h. The cultureswere then washed twice with PBS, fixed with 3% glutaraldehyde, dehydrated, dried, and coated with gold in a Bio-Radsputter coater before imaging. White arrows indicate long fine filopodia. Hatched arrows indicate very flat, thin cellmembranes. Black arrows indicate raised, more rounded, cells.

370 PARKER ET AL.

authors to accommodate the potential adsorption oftracer molecules to the biomaterial. The data pre-sented here also suggest that these materials may beuseful in drug delivery, liquid chromatography, andother adsorption applications. Moreover, we suggestthat these materials may be useful as delivery vehiclesfor specific growth factors for targeted stimulation ofosseointegration of the material in orthopedic applica-tions. Phenotypic evidence suggests the fluorinatedhydroxyapatites may have some effect on the differ-entiation processes of osteoblasts, although this hy-pothesis remains to be tested.

The authors thank Professors Mark Pearcy and JohnEvans and Dr Richard Clegg of the School of Mechanical,Manufacturing and Medical Engineering at QueenslandUniversity of Technology (QUT) for useful discussions; DrDeborah Stenzel of the Analytical Electron Microscopy Unit,QUT, for assistance with electron microscopy; Dr AlexAnderson of School of Life Sciences, QUT, for assistance andadvice regarding the statistical analysis; Professor GraemeGeorge, Dean of the Faculty of Science, QUT; and ProfessorAdrian Herington, Head of the School of Life Sciences, QUTfor useful advice on the manuscript.

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