COLLOIDS.\.\] .S[RFACES

Alot.311..O.J1~\

B_L;~ELSEVIER Colloids 3nd Surlac;:s B: Biointertaces lIt 1998) 131-r~

mi.t:.:iinna. Physico-chemical properties of human plasma fibronectin

binding to well characterized titanium dioxide'-'

~ ~.

.1996)

D.E. MacDonald a.b.c, B. Markovic b, A.L. Boskey c, P. Somasundaran b& V.A. .1.[f?dical Center. Bran.\". ,VY. L'S.-4

b Langmuir Center.for Collouis and Interlaces. Colunlbia University. ,Yew York. .VI~ (.'5.-4C Hospital/or Special Sltrger,v. ",'eI" lork. N'Y. US."

R~ived 19 November 1997; accepted 23 March 1998

s~'\915.

Abstract

~

Introduction to various physico-chemical and bioch~mical int~r-actions bet\\'een the metallic surt'ace and macro-molecules from the soft tissue. as \\'ell as bod~'fluids containing various macromolecules and dis-solved inorganics. Som~ macromolecular speciesare adsorbed rapidly, some desor~d and replacedby other prot~ins. and still others d~nature orfragment once bound [1.2].

Interaction of metallic implant surfaces \vithsurrounding tissues and body fluids is a complexprocess involving different cell types and hostfactors. Upon surgical placement of an implant.the implant metallic surface and the sur2icallyexposed tissue come into close apposition I;ading

132 D.E. ,\(acDonaIJ et aL Co[[oitls Surfaces 8: 8iointerfaces II (1998IIJI-IJ9

2. Materials and methods

2.1, .\.farerials

' 11 r . ... ltanla

Titanium (IV) dio~ide (99.99% pure) wasobtained from Aldrich Chemical (;\Iil\vaukee.WI). The loss on ignition of 0.055% and traces ofcalcium and iron (65 and 5 ppm respectively) werespecified by the manufacturer.

2.1.2. FibronectinHuman plasma fibronectin was obtained in

lyophilized form from Gibco BRL. (LifeTechnologies, Inc. Grand Island, N.Y.). The pro-tein was reconstituted in phosphate butT~red saline(PBS). pH 7.4 and stored at 4"C in plastic vials.

2.1.3. ReagentsPBS was obtained from Gibco BRL (Grand

Island. NY). Sodium nitrate and sodium hydrox-ide (Fisher Scientific) were of ACS reagent grade.

2.2. ,\[ethods

Characterization of titanium dioxide powderswas performed before and after fibronectin adsorp-tion. The characterization methods used were:X-ray diffraction. size determination by light scat-tering, scanning electron microscopy. surface areaby nitrogen adsorption. differential scanning calo-rimetry. and zeta potential by microelectrophoresis.The X-ray diffraction pattern (Phillips 3000 powderdiffractometer, 35 kV. 25 m..\) of the titanium diox-ide was evaluated to deternline the oxide allomorphpresent. Particle diameter was measured using for-~.ard light scattering (l\-[icrotrac Model 7995-10.Leeds and Northrup. Inc.. St. Peterburg. FL) indilute suspension and by scanning electron micro-scopy (Cambridge Stereoscan 250 ~[K1). Thespecific surface area was measured by Brunauer-Emmett-Teller (BET) nitrogen adsorption witha Quantasorb Sorption System (QuantachromeCorp.. Boynton Beach. FL) in which th~ sampl~was first degassed at 300=C for 4 h. Djff~rentialscanning calorimetry ~.as ~ormed on th~ titaniumpowder with a DSC 910S TA Instrum~nt (~~\VCastle. DE) in \vhich 17.77 mg titanium dioxide

The physico-chemical nature of the implant met-allic surface can be expected to influence macromo-Ic:cular binding to it [1.2). In thc: case of titanium.thc: most common metal used in medical and dentalimplants, this interaction is influc:nced by a thin0.5- 7 nm titanium oxide layer [3] which preventsI:orrosion and hydrogen embrittlement [4].. Themain oxide species is TiO: [5). This layerundergoes changes. bc:coming increasingly polar-ized and thicker upon prolongc:d exposure to aphysiological environment [6. i]. Therefore. thisoxide layer plays a major role in protein bindingand cellular interaction.

It has been reported that shortly after implantinsertion, a meshwork rich in fibrinogen andfibronectin forms bc:t\~'een the soft tissue and theimplant surface [8. 9]. Fibron~ctin is a matn.'"{glycoprotein that mediates the attachment of cellsand proteins to each other, as \vell as to the extra-cellular matrices [10). Fibronectin exists in a solu-ble form in plasma and as insoluble cellular formsin association with cell surfaces and extra-cellularmatrix [11.12]. The plasma and cellular fibronec-tins are similar in structure and properties [13.14).Plasma fibronectin is a disulfid~-bonded dimer_\-1, =440000. It is composed of several structuraldomains that are link~d by protease-sensitive pep-tide segments [15-18]. These domains have speci-ficity of binding to cells. bacteria. collagen.glycosaminoglycans. actin. fibrin. fibrinogen. andother biological matt~r [10,16.19].

Because fibronectin is involved in cell adhesionand extra-'Cellular matrix interactions. its physico-ch~mical behavior at implant surfaces is of particu-lar interest. Fibronectin will bind to a wide varietyof artificial substrates and promote cell adhesionto them 120. 21). It has the capacity to bind toboth hydrophilic and hydrophobic surfaces [21-26). Since the nature of protein binding may beaffected by the physico-'Chemical characteristics ofth~ oxide layer formc:d. a better understanding ofthis interaction is important in the developmentand modification of any implanted device.Therefore. the objective of this \vork was to investi-gate the adsorption and desorption mechanisms offibronectin on well characterized TiO! and todetermine changes in oxide particle intertacialpotential as a result of its adsorption.

D.E. .~lacDonuld..r al. Colloids SurJaces B: Bioinrerjaces 111 1998) 131-119

and all contain~rs were prewetted b~fore addingthe protein solution [28].

was equilibrated at 80"C and the temperatureincreased at a rate of 10"C/min to 6QO"C. Zetapotential measurements were performed using0.01 \\"t% of titanium dioxide in 0.03 mol;dmJ NaNOJ with a Pen Kem LazerZee'Mode1501(Bedford Hills. NY). In order to remove anyparticle surface contaminants. several grams of tita-nium dioxide were washed in dilute NaOH solution(0.01 mol/dmJ) using a continuous extractionSoxhlet apparatus. followed by three \,,-ashings \\ithNanopure water (17,5 MQ/cm). Washed sampleswere dried in a vacuum desiccator for 14 h at roomtemperature and stored in stoppered Pyrex glasscontainers to prevent atmospheric contamination.ESCA analyses of unwashed and washed titaniumdioxide samples were conducted using a Perkin-Elmer Physical Electronics model 5600 ESCA s~-trometer. Samples were probed to a depth of 100 A.An analysis area of 800 ~1 was used tor character-ization of all samples.

2.3.1. Adsorption kinetics50 mg of titanium dioxide powder was sus-

pended in 0.5 ml PBS in Eppendorf tubes andequilibrated tor I h on a chemical rotator (FisherScientific) at 25:C. u'I labeled fibronectin (5 ng)in 0.5 ml PBS was added to the titanium dioxidepowder. The samples were then mi.'ted on a rotator(Scientific Industries, Inc.. Bohemia, ~}-) at.roomtemperature for times ranging from I min to 1..1. h.centrifuged at 5000 rpm. and the CPM measuredon a 5236 Packard Auto-Gamma Spectrometer.The supernatant was removed and both the iso-lated supernatant and remaining contents ofEppendorf tubes were counted. To correct foradsorption to the tube walls, the Ep~ndorf tubeswere washed three times with PBS to remo\"~powder, and the tubes recount~d. Binding to tita-nium dioxide was detennined based on th~ l':~Icontent of th~ supernatant and contain~r at eachtime interval. The tim~ intervals for adsorptionwere I, 10. 30, 60 min. and 2. 8. and 1..1. h.Measurem~nts were perfonned in triplicate foreach time point.

~.of

ere

2.3. Adsorption and desorption

Adsorption, desorption, and kinetic experimentswere conducted with radio labeled fibronectin.Fibronectin (0.05 mg) was incubated with 0.5 mCiNa1.!sI (New England Nuclear). oxidized \\ith250 mg;'ml Chloramine T for 10 min. follo\\'ed by96 mg,'ml sodium metabisulfite reducing agent in0.05 M sodium phosphate buffer (PBS) containing1% BSA. pH 7.5, The iodinated matrix proteinpurification was accomplished on a SephadexG50M column (Pharmacia), and the counts perminute (CPM) determined for the eluates ona 5136 Packard Auto-Gamma Spectrometer(Downers Groves. IL). The background CPM\\'ere obtained using control samples of fibronectinin PBS. To insure that l.!sI labeling had no effecton the adsorption properties, labeled and unla-beled proteins were used in different concentrationratios [27]. Within experimental errors no prete-rential adsorption could be detected in theseexperiments. Purity of the labeled preparationswas verified by comparing labeled \\.ith unlabeledprotein by SDS-PAGE electrophoresis. To reducethe effect of adsorption of fibronectin ontothe glass reaction vessels [28]. polypropyleneEppendorf tubes and plastic pipette tips were used.

.ersrp..:re:;3t-rea110-'sis.

d~r

ox-

rph:or-

.10.I in:ro-Th~.~r-.ithIrnelpleilia}urniew\.ide

2.3.2. DesorptionSamples were prepared in the same manner as

for the adsorption assay. After 1 h of incubationwith radio labeled fibronectin. the supernatant \.asreplaced by an equal volume of PBS buffer. Atspecific time intervals (15 min. 30 min. 8 h. 10 h.45 h. 59 h. 69 h). the samples were centrifuged.and the su~rnatant replaced \\.ith an equal volumeof buffer. To account for any interstitial fluidremaining bet\\.een the titanium dioxide particles.the precipitate and Ep~ndorf tubes \vere weighedon a microgram balance at each time inten.albefore and after supernatant removal. Again. allmeasurements were performed in triplicate.

2.3.3. Adsorption isothermThe adsorption isotherm of human plasma

fibronectin on the oxide was determined by sus-pending 50 mg titanium diox-ide powd~r in 0.5 mlPBS and equilibrating tor 1 h at room temperatureusing a tu~ rotator (Scientific Industries. Inc..

~~_. ~-~~...~ ',.,---

].a DE. .\I&1cDotk,ld..r &lL Colloids Su~faC#!s B: Bioillr..~,;,ces II i 1991. 'JI-1J9

Bohemia. ~"). Each sample was then incubatedwith 0.5 ml of a solution containing fibronectin inPBS (0.0750 to 1.800 mg ml, and ~quilibrat.:d t-orI h on the rotator at ~5'C. The samples werecentrifuged at 500) rpm and the supernatant col-lected for protein analysis. The pH of the sampleswas measured as pH 7 .~. [nitial and residual con-centrations of fibronectin in solution wer~ mea-sured using a total organic carbon analyzer(Dohnnann DC90). To detennine the extent ofprotein binding to the polyetbyl~ne tubes. triplicatecontrol sampl~s (without TiO~ po\\'dc:r) were runin parallel and analyz~d using a BioRad proteinassay (Bio-Rad Laboratories. Hercules. CA). Theprotein assay allowed the determination of anyprotein tbat may bave adsorbed to tbe reactionvessel despit~ tbe careful protocols followed.Protein adsorption density. r, was calculated fromdepletion values. The parking area per moleculewas calculated from the adsorption plateau valueand the particle surface area as detennined by theBET tecbnique and using a molecular weight of440 000 [19] for the protc:in.

Fig. 1. s.::lnning d~tron mkrograph or titanium dio~i,je. magnification ~OOO x .

>-e..~

I~

2.3.4. Ele;..trokinetic stlldiesZeta potential of both titanium dioxide powder

and titanium dioxide powder reacted ~ith HPFwas determined by m~asuring the particle electro-phoretic mobility using a PenK~m Lazer Z~~Model 501 (Bedford Hills. ~'). pH i .~.

pH

FiJ. ~- Zeta potential ot' til3n1um di,,~id~ ~i..'re and 1j:~r~-a1!Iin@ (/81)1>3 m"t dmJ Na~O,1 - m~3n=$D-3. Results

X-ray diffraction analysis showed the presenceor only on~ crystallin~ TiO1 phase. rutil~. Themean TiO.: particle diameter was 7."::t3.3 nm byforward light scattering with a Microtrac System.Scanning electron microscopy also sho\~'ed a largenumber or $maller particles ( < I J.UD) not registeredby the ~(icrotrac Syst~m (Fig. I). The particlesurface ar~a was mea$ured to ~ 2.3 m': g u$ingthe BET m~thod.

A comparison or z~ta pot~ntial of titaniumdioxide at different pH values for both unwashedand washed uncoated $amples v."ith 0.01 moljdmJNaOH ( Fig. 2) shows th~ un".ashed titanium diox-ide to exhibit two isoelectric points (5.2 and 6.4)

\vhil~ th~ wash~d sho\\"~d one at 6.~. Th~ un\\-ash~dsample was n~gative at values ~Iow pH 5 \vhilethe \\-ashoo samples \\"ere positive.

Differential scanning calorimetry (DSC)detect~d no bulk contaminants at t~peratures upto 6OO'C (r~sults not sho\\-n). How~ver. ESCAanalysis of un\\"ashed and \vashed titanium dioxid~(survey sIX'Ctra giv~n in Fig. 31 sho\voo \\"3shingto reduce th~ amounts of silica. organic nitrogen.and chloride species on the sufi-ace I. Tabl~ II.

Adsorption of radiolabeled fibronectin on tita-nium dioxide occurrc:d rapidly \\"ith ma.'timumbinding in less than 10 min and a d~finit~ decre~at longc:r tim~s (Fig. ~ I. The per~('ntage adsorbed

DE. .\/ucDollald t!/ al. Colloids Su,:laces B: Bioiruer!aces f f (/998) /3/-/J9

ao

~

)""'~~

]

J3G'"r

~

t~.~CI»

S10

0 f .,. I0 I 2}'" 6 7

WuhN..o..

Fig. S. Desorption of human plasma fibronecun :rom titaniumdioxide by subsequent washing in PBS - me:ln:::SD. The }.-a.~ shows the ~rcentageof fibronectin remaining as a functionof wash num~r.

Table IESCA atomic concentration table for unwash.:d titanium diox-ide (TiO:) samplc and washcd ~ith 0.01 N NaOH and nano-pure waterFig. 3. ESCA survey spectra of titanium dioxide ~ft)re la) and

after (b) washing. Concentration (at. cc)Element

Un,,'ashed lilaniu,', diQ.~ide

C.S

0.,

Ti1p

Si:.

N.,

C':.

18.7656.7521.7\

1.13O.~5O.:!O

Tilaniw', .lio.\"1.1.. \,.ashed ',ilh .VuOH un.1 tlalwpllr.. ,,'ut..rC10 \4.i7°'0 ;9.,",TiZp 24. isSiZp 0.60N,o 0.3\CIZp 0,\3

C)up:Aid~.ng

0 lO .ao fA) 80 100 IlO 1000Tame.min

Fig.~. Kinetics "f human plil$tn;1 fibr"n«tin adsorption ontitanium Ji"xide (in PBS. pH 7.~ I - mean::: SO. Th~ .r-axi$$h,,\v~ the percentag~ ",- fibron«tin :1J$orbeJ ;1S :1 function oftim~.

tion in adsorbed protein occurred during the initialbuffer washings (Fig. 5) with minimal subsequentremoval.

Evaluation of the adsorption isoth~rm ot- unla-beled fibron~ctin onto titanium dioxid~ showed aninitial slo~ suggestive of a moderate affinity. Th~isotherm d~monstrated a plateau value of0.21 mg;;m2 <. Fig. 6) and the plateau value foradsorption density corresponds to a fibronectinmonolayer. From the adsorption pla[~au. th~

is defined as that percentaget of initial protein insolution which binds to the oxide particle underthe experimental conditions. Desorption ot- fibro-nectin from the particles by a series of butTerwashings over time showed that a significant reduc-

ta-urnase~

.:.of'

136 D.E. .~/acDonald t!r al. ColJo;(u Surfact!s B: Bioinrt!,:faces I J (1998) 111-/.19

~

t;:

a}

f

1°1

100 :00 300Rcsaiual~. ppm

.aoo .Fig. 6. Adsorption isotbenn ,,( human plasma fibronectin ontitanium dioxide (in PBS. pH 7.4) - mean:tSD.

~

at

~;"015

I"a

10.15

~<

0.05

0

Fig. 7. Adsorption isotherm I)j human plasma fibronectin ontotitanium dioxide and corr.:sponding panicl~ zeta potCQtial afteradsorption (in PBS. pH 7~) - mcan=SD.

rapidly at physiological pH. Desorption occurs\vith repeated washings, but th~ adsorption processis not fully reversible. Thes~ results support theinterpretations that fibronectin has a mod~rateaffinity for titanium dioxide.

The surface of the powders used for the adsorp-tion studies was, as previously reported [30,31],slightly anionic, and based on ESCA data. showedthe presence of impurities of silica, organic nitro-gen, and chloride species. These impurities canresult in a shift in isoelectric point (iep) of theoxide [30]. The anionic contamination may beattributed to the presence of TiCI4. It has beenreported that titanium dioxid~s show iep valuesranging from 4.6 to 6.4 due tb variations in pre-treatment heating, washing protOCols, atmosphericinteractions, and method of storage [3l,32}.

The binding of fibronectin to the titanium diox-ide powder was comparable to that report~d forits binding to germanium [33]. This is not surpris-ing since germanium also has a hydrophilic surfacesimilar to titanium dioxide [33-36} and sinceadsorption is determined in this case more by thesurface of the adsorbate than by the nature of theadsorbent [37]. In this study only 57% of fibronec-tin adsorbed (Fig. 4), possibly due to the hydro-philic nature of the particle surface [38].Fibronectin is known to adsorb to a greater ~xtenton hydrophobic rather than hydrophilic surfaces[28,39}. Similar binding characteristics ha\'~ beenreported for other serum prot~ins on similar sur-faces [~0-43].

This study focussed on binding to \\"elt charac-terized titanium dioxide ~cause th~ ch~micalnature of the solid surface determines th~ modeand strength of binding and conformationalchanges of the adsorbed prot~in [37]. Antibodybinding to adsorbed fibron~tin sho\ved moreexposure of antigenic sites when the prot~in isbound to hydrophilic than onto hydrophobic tissueculture plastic [44]. Such surface depend~nt con-formational changes have b~en observed by ellip-sometry [39,45} and total internal red~ctionfluorescence spectroscopy [~6]. Heparin and cellbinding fragments of fibronectin become moreexpo~ed after binding to pretreated heparinizedand nonheparinized poly (vinyl chloride) surfaces.suggesting protein orientation)o be surface and

calculated parking area per molecule was3.48 x 103 nm::.

A comparison of the zeta potential to theadsorption isoth~nn shows a good correlationbetween the increase in adsorption density offibronectin on titanium dioxide and the increasein negative zeta potential <. Fig. 7).

4. Discussion

Results in this paper indicate that the initialfibronectin attachment to titanium dioxide occurs

D.E. .VacDonald et aL Colloids Surfuces B: Bioillterfaces II (1998) 131-139

.t'

il].ved

ro-

:anthe

~eenues)re-eric

IOX-

forlriS-faceincethe

.thenec-jro-

38).tentaces:>eensur-

hydrophobic polystyrene [~.65J. The presence 1.)[a negative surtace potential of titanium dioxidc:may also influence desorption during the washingcycles. Desorption studies of adsorbed cytochromemutants (E 15Q and E48Q) using butter washesfound protein preadsorbed to negatively chargedsurfaces (stearic acid) were fully reversible. Onlyon highly positive (polyallylamine) and neutralsurfaces (POPC) was there minimal reversibilit~-[53 J. As in the case of adsorption. a small variationin amino acid composition can affect desorption[52J.

The slope of the adsorption isothenn (Fig. 6)indicates a moderate affinity of the protein for thetitanium dioxide surface [37]. Ellipsometric workon fibronectin binding to modified hydrophobicand hydrophilic silica has reported the binding tobe 2.74 and 2.45 mg/ml respectively with a plateauvalue concentration of 20-30 ~g:!ml [39J.Differences between the values in the literatureand our results could be due to the differentsubstrates employed [65,66]. The surt'ace concen-tration at the fibronectin isotherm was lower thanthe expected value based on molecular size. Thishas been observed in other protein-surface systemsand can be explained by the fonnation of a lessdense monolayer and unfolding of the molecule

[37].Zeta potential measurements showed the iep of

the washed, hydrated titanium dioxide to be 6.2.and it has \\.eakly negatively charged surface(-16 mV) under physiological conditions (Fig. 2).These values are in the range of previously pub-lished data [32.67,68]. The iep of fibronectin is5.5-6.3 [13,69-71] and under the experimentalconditions (pH 7.4) it would carry the overallnegative charge as well. Ho\vever. it is to be notedthat different regions of the protein are markedlydifferent from each other in terms of acidic andbasic properties [70.72.73]. Hence. the proteinadsorption process can be driven by the electro-static interactions bet\veen the positive sites of theprotein and the negative particle surface sites. Theobserved increase in negative zeta potential afterprotein adsorption (Fig. 7) can be accounted forby the tertiary geometric orientation the proteincan assume during the process of protein binding.Fibronectin is known to assume different geometric

precoating dependent (47]. Intersubunit distanceof two Gln3 residues increases (48]. two freesulfhydryl groups (SHI and SH2) show positionalchanges (49.50]. and two amino-tenninal regionsbecome separated upon surface binding to Cytodexbeads [51]. an verifying that fibronectin undergoesconformational changes during binding.

Another important factor in protein adsorptionis related to the nature of the protein itself. Twohomologous serum proteins from dissimilar species(bovine and human albumin) showed considerableadsorption differences when tested on a silica-titania surface [52]. These results show that theadsorption behavior of two serum proteins isgreatly influenced by their respective amino acidsequences. Moreover, single point mutations havebeen shown to have a major impact on proteinadsorption on a variety of Si(Ti)Oz surfaces withpositive. negative. and neutral charged coatings[53].

The slight decrease in attachment of fibronectinto the titanium dioxide after I h (Fig. 4) may berelated to protein-particle surface concentration.Protein unfolding. as observed with atomic iorcemicroscopy, decreases with increasing particle sur-face coverage [54-57]. This decrease in proteinstructural rearrangement and unfolding results ina reduction of potential exposed binding sitesreducing surface adsorption [58-60].

Fibronectin on titanium dioxide showed a par-tial desorption profile (Fig. 5) similar to hydro-philic silica [39]. Protein adsorption has beendescribed as a nonequilibrium irreversible processbecause many proteins do not completely desorbfrom metal oxide surfaces [2,45,60,61]. Some irre-versibility may be due to the protein's tendency todenature over period of time [62,63]. Specificbinding activity is related to protein size due tomultiple protein contacts on a surface [55].Simultaneous rupture of most of these bonds at agreater number of sites would be required to totallydislodge the protein. Since fibronectin is a largemolecule with numerous binding sites. this mayexplain why desorption was not completely revers-ible [42,64]. Surface properties should certainlyinfluence protein desorption in the same manneras the influence adsorption. Fibronectin, for exam-ple. is more easily removed from hydrophilic than

lrac-.tical\odeonal

>Odynorein isiSSuecon-

~lIip-:tion1 cellmore:lized'aces.

and

-- -" , .,It""-

D. E. ,\./aC'DlJnald..1 al. Colloids Szzrjacl!s B: Bioinl..,:1JC'l!s 11 ,t99S IJl-IJ9

sha~s de~nding on the particular surface it bindsto and the conditions under \vhich it has beenpreserved [33.57.7+-77). The degree with whichcontonnational changes occur are also proteinspecific. Adsorbed Factor XII [78] and molecularIgG protein binding is reported to occur withextensive unfolding [79). Circular dichroism [70)and elJipsometric studies [55,80.81] of fibrinogen.prothrombin. and serum albumin. show little orno rearrangement of the adsorbed layers. Asidefrom the different surfaces used. ex~rimental con-ditions may also influence protein contonnation.Plasma fibronectin in solution has been describedas an extended structure under acidic. alkaline, orunder conditions of high ionic strength when visu-alized by rotary shadowing [74.75]. The use of30% glycerol in the sample preparation of fibro-nectin for electron microscopic observation ofrotary shado\\'ed specimens is sufficient to yieldextended structures [48.76,77]. In the absence ofglycerol, the protein assumes a compact roundedstructure.

be possibl~ to improve cell binding ability ot-implantable materials.

Ackno,,-Jedgment

The authors acknowledge the support of th~Physicians Career Development A\vard. V.A.Central Office. NIH Grant No. DE04141 and NSFGrant No. EEC-940~989. Special thanks go to Of.Michael Stranick (Colgate-Palmolive Company-Piscataway. NJ) for the ESCA analysis.

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5. Conclusions

Studies of plasma fibronectin binding to a wellcharacterized implant material oxide have pro-vided insights into the ways in which such materialsmight be manipulated to optimize their in vivoperformance. Fibronectin was shown to bindrapidly to titanium dioxide with a moderate affin-ity. The measurement of fibronectin binding totitanium dioxide yielded an isotherm with a plateauvalue of 0.21 mg/m: at an equilibrium concen-tration of 0.4 mgjml at pH 7.4. Fibronectin coatingof oxide particles caused an increase in its negativezeta potential, partly as a result of electrostaticinteraction between positively charged proteingroups and the negatively charged particleexposing negatively charged protein groups at theftuid-protein interface. These results in a simple invitro system suggest that by altering the implantoxide surface, it may be possible to manipulateprotein binding in vivo. In doing this. by alteringthe properties of the bound protein such that cellbinding domains are optimally presented. it may

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