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Laboratory Investigations

Effects of Roughness, Fibronectin and Vitronectin on Attachment,Spreading, and Proliferation of Human Osteoblast-Like Cells (Saos-2)on Titanium Surfaces

I. Degasne,1 M. F. Basle,1,2 V. Demais,1 G. Hure,3 M. Lesourd,2 B. Grolleau,4 L. Mercier,1 D. Chappard1

1LHEALaboratoire dHistologie-Embryologie, Faculte de Medecine & CHU Angers, rue Haute de Reculee, 49045 Angers-Cedex, France2Service Commun de Microscopie Electronique, Universite dAngers, Angers, France3Private Practice, Paris, France4Laboratoire des Plasmas et des Couches Minces, Institut des Materiaux de Nantes, Nantes, France

Received: 14 November 1997 / Accepted: 1 November 1998

Abstract. The influence of surface roughness and the pres-ence of adhesion molecules in the culture medium werestudied regarding cell adhesion, shape, and proliferation ofosteoblast-like cells grown on two types of titanium disk.Type I disks were acid etched and type II disks were sand-blasted and acid etched. Surface roughness was determinedby contact profilometry and scanning electron microscopy.Chemical composition and oxide thickness of the superficialtitanium layer were established with energy dispersive X-

ray spectrometry, electron spectroscopy for chemical analy-sis and auger electron spectroscopy. Titanium release in theculture medium was assessed by inductively coupledplasma-optical emission spectrometry. Osteoblast-like cells(Saos-2) were cultured on both types of titanium disks (1) instandard conditions (DMEM culture medium supplementedwith fetal calf serum), (FCS), (2) with the culture mediumalone (DMEM alone), (3) in the presence of fibronectin orvitronectin (DMEM supplemented with fibronectin or vit-ronectin). Cultures were also performed in the presence ofmonoclonal anti-integrin (

1,

v) to test the cell adhesion

molecules involved in the cell binding to the titanium sur-face. We found that sandblasting does not modify the

chemical surface composition and that titanium representsonly 56% (in the atom percentage) of surface elements.Release of titanium in the culture medium was found toincrease from 24 to 72 hours. In the absence of FCS, fibro-nectin, or vitronectin, cells appeared scanty and packed inclusters. On the contrary, cells cultured in the presence ofFCS, fibronectin, or vitronectin were flattened with largeand thin cytoplasmic expansions. The addition of anti1orv

integrin subunit monoclonal antibody in the culture me-dium decreased adhesion and spreading of cells, particularlyin the presence of fibronectin. Cell proliferation was sig-nificantly higher on culture plastic than on both types ofdisks, but was increased on rough but not on smooth sur-

faces. These results indicate that a high surface roughnessand presence of fibronectin or vitronectin are critical ele-

ments for adhesion, spreading, and proliferation of cells ontitanium surfaces.

Key words: Osteoblast Titanium Fibronectin Vitronectin Roughness Integrins.

Titanium (Ti) has become one of the most commonly useimplant material in oral and cranio-facial surgery [1, 2Several factors have been demonstrated to influence thinterfacial response of bone cells to the implanted titaniummaterial, including biocompatibility, surgical technique, design, and surface status (morphology and chemical composition) of the implant [3]. The functional activity of cells icontact with the implant surface is expected to be determined by the properties of the implant surface [4]. Amongthem, surface topography, roughness, and Ti oxide thickness have raised many studies [58]. The overall picturethat emerge from in vitro and in vivo studies is that osteoblasts attach and spread more readily on rough surfaces thanon smooth ones. Although Ti has been used extensively aimplant material in different medical applications for ove30 years, factors and mechanisms underlying the biologicaresponse to Ti are poorly understood. A mineralized extracellular matrix was reported being elaborated in vitro bosteoblasts and cells derived from rat bone marrow, cultureon Ti [9, 10]. Processes by which cells become establisheupon a surface involve initial attachment followed by cellular spreading. The molecules involved in cell adhesioand spreading include extracellular matrix (ECM) molecules, transmembrane receptors such as integrins, and intracellular cytoskeletal components [11]. Cellular interactions with ECM molecules are believed to generate specifisignals that are transduced through the integrins to the cy

toplasm, the cytoskeleton, and the nucleus [12]. Severaextracellular matrix proteins such as collagen, thrombospondin, fibronectin, vitronectin, and osteopontin, some owhich are present in serum, have been shown to mediate celattachment to substrates. Among them, fibronectin and vitCorrespondence to:M. F. Basle at the LHEA

Calcif Tissue Int (1999) 64:499507

1999 Springer-Verlag New York Inc



ronectin are proteins found in many extracellular matricesand in blood plasma. They promote cell adhesion and reor-ganization of the actin microfilaments [13], thus influencingthe cytoskeletal network, cell morphology, and migration.The contribution of the serum fibronectin and vitronectin tothe attachment and spreading of cells is estimated differ-ently from one study to another [14, 15].

The aim of this work was (1) to investigate how differentsurface roughness may influence cell adhesion, shape, and

proliferation of osteoblasts and (2) to determine the role offibronectin or vitronectin and of their specific cell ligands(

1and

vintegrins). Titanium surface roughness was de-

termined by profilometry and scanning electron microscopy(SEM). Chemical composition and oxide thickness of thesuperficial titanium layer were established with energy dis-persive X-ray spectrometry (EDX), electron spectroscopyfor chemical analysis (ESCA) and auger electron spectros-copy (AES). Saos-2, a human osteoblast-like cell line, wascultured on two types of titanium disks with different rough-ness. The cell morphology was studied by SEM and tritiatedthymidine incorporation was used to assess cell prolifera-tion.

Materials and Methods

Titanium Disks

Commercially pure (cp) grade 3 titanium disks (8 mm in diameter,2 mm thick, 445 mg) were cleaned by sonication in a detergentsolution (Micro 12, SPAD, France) for 20 minutes and rinsed bysonication in distilled water for 20 minutes. Two types of diskswere then prepared. Type I was not sandblasted but received nitricacid passivation treatment for 15 minutes. This treatment was re-ported to produce a more inert surface by increasing the thicknessof the superficial oxide layer [16, 17]. Disks were sonicated for 20

minutes in the detergent solution and for an additional 20 minutesin distilled water. Type II disks were sandblasted with silica par-ticles, 225550m in diameter, blown under 23 bars of pressure,acid etched (nitric acid 52%, hydrofluoric acid 10%, in distilledwater) for 5 seconds and sonicated in the detergent solution anddistilled water as above. Disks were then processed for 10 minutesby radio-frequency glow discharge (Picotron, Park Dental Re-search Corp., NY) to achieve the cleaning [18] and sterilized by irradiation (25 K. Greys) in a polyethylene terephtalate box sealedwith a Tyvek sheet (Dupont de Nemours).

Surface roughness of titanium disks was assessed by contactprofilometry [19] (Talysurf 10, Rank Taylor Hobson). A 2.5 mdiamond stylus was used to determine the centerline averageroughness values (Ra) along a 2.5 mm length. Ten individualmeasures per disk were made on five disks for each type of surfacetreatment. Ra was determined as the mean value (SD) for allmeasurements.

Surface morphology was studied by SEM, using a field emis-sion scanning electron microscope (Jeol JSM-6301 F) at 5-KeVacceleration voltage. For five disks of both types, six micropho-tographs were taken at a magnification of 3000 and were used todetermine the roughness index on a Quantimet Q570 image ana-lyzer (Leica) with an algorithm previously described [20, 21].Briefly, the SEM images were numerized with a Sony Tri-CCD910P camera in 256-gray-level images. For each gray image, sev-eral binary images were obtained with successive thresholds sepa-rated by 30 gray levels. So, for a given gray image A, a new imageA

was obtained for each threshold. For each A

, a new binaryimage BA

was obtained after boundary identification, and the

number of pixels was then measured. The composite image wasthen obtained by making BA BA

+1 . . . BAn, and theroughness index (RI) was defined as follow: RI n (BA

)/Ar,

where Ar represents the whole surface area.The elemental composition of the surface of both types of disk

(n five per type) was determined by EDX (Link-Oxford), with

a depth analysis of about 1m, and by ESCA (LHS 12 Leyboldusing a Mg anode, with a depth analysis of about 5 nm. Oxide Tthickness at the surface of both types of disks was estimated bAES (LHS 12 Leybold) at 5 KeV with argon sputtering (1 A) [78]. The erosion rate of SiO2from a Si single crystal wafer was useas reference.

Cell Culture

Osteoblast-like cells (Saos-2, ATCC HTB 85) were cultured iDulbeccos Modified Eagles Medium, DMEM (Eurobio), supplemented with 0.4 mM/100 ml L glutamine (Eurobio) and 10% FC(Eurobio). Cultures were maintained at 37C in a humidified incubator in the presence of 5% CO2 and subculturing was performed using a 0.01% trypsin solution in phosphate-buffered saline (PBS), pH 7.4.

Titanium disks were placed in 24-well plates, one per welCells were carefully seeded at 105 cells in 50 l, allowed to adherfor 2 hours, and 1 ml of medium was then delicately added to eacwell. Cultures were carried out for 24, 48 or 72 hours under fouconditions: (1) DMEM supplemented with 5% FCS (standard medium), (2) DMEM alone, (3) DMEM supplemented with 2g/mof fibronectin from human plasma (Sigma), and (4) DMEM

supplemented with 2 g/ml of vitronectin from human plasm(Sigma). Furthermore, cultures were also carried out in the presence of monoclonal antibodies directed against1(CD 29, Dakoorv (CD 51, CLB) integrin subunits.

Titanium Release

Titanium disks were incubated in 24-well cell culture plates wit1 ml of the standard medium. Titanium release in the supernatantwas determined by inductively coupled plasma optical emissiospectrometry (ICPOES) (Jobin Yvon 48, Longjumeau, France) fofive samples of type I and type II disks and for five well control(standard medium without titanium disks), at 24 and 72 hours.

Scanning Electron Microscopy

SEM was used to study the morphological characteristics of cellin culture. Each type of culture was performed in triplicate (foeach surface treatment, time of culture, and type of culture condition). Cultured disks were fixed in 2.5% glutaraldehyde in phosphate-buffered saline (PBS), dehydrated in graded ethanol seriestreated by hexamethyldisilazane (Sigma), and air dried. Samplewere coated with carbon by sputtering (MED 020, Bal-Tec) beforSEM examination (Jeol JSM-6301 F).

Cell Proliferation

Cells were seeded at 105 cells in 50l onto both types of titaniumsurfaces and on control plastic dishes (24-well cell culture) (Costar) and completed to 1 ml after 2 hours with the standard mediumCulture wells were placed at 37C in a humidified, 5% CO2 amosphere for 12 hours to equilibrate. The medium was then replaced with a fresh standard medium containing 0.5 Ci ml

tritiated thymidine (methyl-3H) (Amersham) for further 24 and 4hours. Five samples from each surface roughness at each time wertested. Proliferation was then assessed as 3H thymidine incorporated into total DNA. Briefly, labeled DNA was precipitated bperchloric acid (HClO4, 0.6 N) and filtrated on a Whatmann GF/Cfilter. Tritiated thymidine was determined with liquid scintillatiocounting (Minaxi Tri-Carb, Packard).

Statistical Analysis

Statistical analysis was done with Systat software, release 6.0.

I. Degasne et al.: Osteoblast-Like Cells in Culture on Titanium500



with nonparametric tests. Kolmogorov-Smirnov and Kruskal-Wallis analysis of variance were used to compare cell proliferationand titanium release, the Students-ttest to analyze surface rough-ness, and the Mann Whitney U-test to compare the chemical sur-face composition.

Results

SEM Surface Topography and Roughness

The SEM appearance of the surface was different in the twotypes of Ti disks. Non-sandblasted type I disk showed arelatively smooth surface, with regular and circular linesdue to machining preparation (Fig. 1A). Sandblasted type IIsurface was highly heterogeneous with many irregular pits,indentations, and ridges (Fig. 1B).

The roughness values calculated by profilometry wereRa 0.30 0.01 SD for type I (n 50) and Ra 0.94 0.09 SD for type II (n 50), (P< 0.001). The roughnessindices calculated by image analysis were RI 0.18 0.05

SD for type I (n 30) and RI 0.30 0.02 SD for typeII (n 30) (P< 0.001). There was a significant correlation(r 0.93,P < 0.01) between the two methods of roughnessmeasurement.

Surface Composition and Ti Release

X-ray dispersive spectroscopy study of cp Ti disks revealedonly the presence of titanium (Fig. 2), whereas ESCA analy-sis demonstrated the presence of contaminants such as car-bon (C), oxygen (O), and silicon (Si) in addition to Ti (Fig.3). The relative elemental concentrations obtained from

ESCA are summarized in Table 1. Differences in the atomicratio for O, C, Ti, and Si were not significant between bothtypes of surface. Titanium was predominantly in the form ofTiO2, as shown by peak energy (458 eV versus 454 eV forpure titanium). The O spectrum was complex due to its

binding to Ti, C and Si. Carbon was in the form of C-C (284eV) and hydrocarbon (285 eV) and Si was in the form of Soxide (103.4 eV versus 99 eV for pure Si). Si was alsodetected in the sealing sheet of the packing. Oxide thickneswas 9.7 nm for type I and 8 nm for type II Ti disks (Fig. 4)

The Ti release was assessed in standard medium cultureafter 24 and 72 hours of culture, by inductively coupleplasmaoptical emission spectrometry. Five samples fromeach type I and type II disks, and from plastic controls wermeasured at each time. The results are summarized in Tabl2. The Ti release was significantly higher for sandblastedisks than for non-sandblasted samples at each time of culture (P< 0.001) and the release significantly increased from24 to 72 hours (P < 0.001).

Morphology of Cells in Culture

In standard medium, cells showed a different morphologica

Fig. 1. SEM of titanium disk surface. (A) The surface of non-sandblasted disk appears relatively smooth and striations are related tmachining. (B)The surface of sandblasted Ti disk appears irregular and heterogen with pits, indentations, and sharp ridges (bar 10 m

Fig. 2. Energy dispersive X-rays spectrometry (EDX). TypicaEDX spectrum observed on all titanium disk surfaces. The profilshows the characteristic peaks of Ti corresponding to specific energy of rays LI, Ln, L, L, K, K.

I. Degasne et al.: Osteoblast-Like Cells in Culture on Titanium 50



aspect on each type of disk. On type I disks, cells wereflattened, displaying a large and thin cytoplasmic layer andnumerous filopodia extending from the cell body to thetitanium surface (Fig. 5A). On type II disks, cells showed asimilar general appearance but were slightly less spread.Some of them had a globular appearance (Fig. 5B). On bothtypes of disks no difference was observed between 24 and72 hours. The addition of anti 1 or v integrin subunitmonoclonal antibodies did not significantly modify adhe-sion and morphology of the cells. However, when culturedin the presence of both antibodies, cell adhesion appeareddramatically decreased.

In the culture medium without FCS, cells displayed asimilar appearance at 24 and 72 hours on both surfaces.They were packed and piled up, often globular in shape, andwith large areas free of cells on the disk surface (Fig. 6A& B).

In medium supplemented with fibronectin or vitronectinbut without FCS, cells were flattened and showed similardisposition than cells cultured in the presence of the stan-dard medium (Fig. 7A & B).

In the fibronectin containing medium, the addition ofanti 1integrin subunit decreased cell adhesion and modi-fied the morphology of cells that appeared globular andpiled up. When an anti-vintegrin subunit was added in themedium instead of anti-1, cells were numerous but dis-played a globular shape.

In the vitronectin containing medium, cells were numer-ous and flattened in the presence of anti

1but scarce and

globular in the presence of anti-v integrin subunit.

Cell Proliferation

Cell proliferation was monitored by 3H-thymidine incorporation, and absolute values in dpm/well are given in Table 3There was no difference between cells grown for 24 houron Ti disks of both types and cells grown in plastic dishesIn contrast, 3H-thymidine incorporation was significantlhigher in cells grown for 48 hours in plastic dishes as compared with cells grown on both types of Ti (P 0.01)Although the level of 3H-thymidine incorporation was similar at 24 and 48 hours for cells grown on smooth surface(type I disks), a significant increase was observed for cellgrown on the rough surface after 48 hours (P 0.03).

Discussion

Titanium in its cp form is reputed to give excellent resultwhen implanted in bone and is now commonly used fodental implants. The quality and intensity of bone cell response to a Ti implant appears to depend on factors linketo the nature of the material and on the ability of bone cellto interact with the implant surface and microenvironmen[4]. Microenvironment is mainly influenced by surfaccharacteristics that can modify cell activity, cell differentiation, bone apposition rate, quality of the newly formed boneand interface contact between bone and implant [22, 23]Several factors (chemical composition, Ti oxide thicknesssurface roughness, and degree of Ti release) have been emphasized to characterize the implant surface [3].

In our study we have examined the implant surfacroughness using two methods. One was contact profilome

Fig. 3. Electron spectroscopy for chemical analysis (ESCA).Typical ESCA spectrum observed on all titanium disk surfaces.Energy peaks of O, Ti, C, and Si are characteristic of titaniumoxide (TiO2), carbon-carbon, hydrocarbon, and silicon oxide.

Table 1. Relative elemental concentrations (in atom %) at thesurface of smooth (type I) and rough (type II) titanium disks(ESCA analysis)

ElementGroup I(n 5)

Group II(n 5)

Ti 5.9 1.6 4.8 1.2O 36.1 3.3 37.7 1.9C 46.3 5.1 45.1 3.7Si 11.7 0.5 12.4 0.8

Average value (mean SD) acquired from five points for eachsurface treatment

Fig. 4. Auger electron spectroscopy (AES). Representative AEdepth profile observed on all Ti disks. Relative concentration of Tincreases and of O decreases in relation to progressive erosion othe surface. Oxide thickness was 9.7 nm for type I and 8 nm fotype II Ti disks.

Table 2. Ti release in medium culture (ng/ml SD) from smoot(type I) and rough (type II) titanium disks

Controls Type I Type II

24h (n 5) 0 (n 5) 7.7 3.8 (n 5) 29.3 572h (n 5) 0 (n 5) 17.3 0.6 (n 5) 50.0 7.

Type I versus Type II: P < 0.001 at any time and 24 hours versu48 hours: P < 0.001 in both types




try and the other was derived from image analysis algo-rithms [20, 21]. Both methods demonstrated that sand-blasted disks had higher roughness indices than non-sandblasted disks and the methods were significantlycorrelated. The chemical composition of the implant surface

was studied by EDX, ESCA, and AES. EDX detected onlyTi (with a 1 m penetration depth) and does not appear tobe an adequate or sufficiently sensitive method for deter-mining the exact chemical composition of the superficiallayer. With ESCA and AES (with a 510 nm penetration

depth), Ti, but also O and C were detected but their relativconcentrations were not significantly different in both typeof disks. ESCA and AES appear to be more sensitive anappropriate methods, showing that the main elements on thimplant surface were carbon and oxygen. Titanium repre

sented only 56% of all elements detected in the superficialayer of the disk surface. The titanium oxide thickness (assessed by AES) was similar in both types of disks, about 10nm, and was of the TiO

2type as demonstrated by the pea

energy. The presence of Si in both types of Ti disks wa

Fig. 5. SEM of osteoblast-like cells in culture (DMEM supplemented with 5% FCS) on (A) non-sandblasted titanium disk surface; cellare flattened with numerous filopodia and microvillosities and(B) on rough sandblasted Ti disk surface. Cells appear less flattened thaon smooth surface. Some globular cells are visible (bar 10 m).

Fig. 6. SEM of osteoblast-like cells in culture (DMEM alone) on (A) non-sandblasted titanium disk surface (bar 10 m) and(B) orough sandblasted Ti disk surface. Cells are packed and piled up in large clusters (bar 10 m).




attributed to the presence of Si in the sealing sheet of thepackage. This result emphasizes the importance of thesample packaging. Thus, both types of Ti surfaces weredifferent only in term of roughness but not in the chemicalsurface composition or oxide thickness.

When placed in standard biological conditions (culturemedium supplemented with FCS), release of Ti was signifi-cantly increased from the roughest surface. These results areconsistent with in vivo local release of Ti. In experimentalconditions (grade 1 Ti fibers implanted in tibiae of rabbits)it was demonstrated that Ti levels in the bone near theimplant raised whereas Ti levels in serum and urine did notincrease up to 1 year after implantation [24, 25]. Moreover,an in vitro report showed that nitric acid passivation doesnot affect the oxide thickness or the trace element releasefrom cp Ti in culture [16]. Titanium ions (in concentrationranging from 0.01 to 100 ng ml1) did not determine dam-age (in terms of cell viability or cell injury) but could inhibitphytohemagglutinin-induced T-cell and liposaccharide-induced B-cell proliferation [26]. They also reduce TGF and enhance IL1 , IL6, and TNF production by ratperitoneal macrophages or human blood mononuclear cells[27, 28]. The activity of glucuronidase, acid phosphatase,lactate dehydrogenase, and glucose 6-phosphate dehydroge-nase were found to be activated in murine peritoneal mac-

rophages after exposure to various Ti concentrations [29]. Iis likely that the release of Ti ions affects cell activities anregulation. In our study, the increased Ti release observewith the roughest surface may be considered the consequence of an increased interface or the consequence of modified status of the Ti superficial layer, or as a combination of both. However, similar chemical surface composition in terms of elements, oxide type (TiO2), and oxidthickness suggests that the Ti release depends mainly on thextend of the surface and thus of the roughness.

Most studies concerning the influence of the implansurface have pointed out the important role played by certain degree of roughness [7, 8, 19, 3032]. Significantlyhigher bone-to-metal contact was found for implant sandblasted with 25 m particles compared with those sandblasted with 250 m particles [33]. Results obtained witsmooth implants suggest that a reduced surface roughness iassociated with a lower rate of bone formation in rabbicortical bone [7]. Inversely, the extent of bone implant interface is positively correlated with an increasing roughnesof the implant surface [5]. Surface roughness determines thtissue reaction at the interface and directly influences thcell activities [32]. Surface roughness also affects chondrocytes and osteoblast proliferation, differentiation, and matrix synthesis in vitro [23]. Furthermore, it was demon

Fig. 7. SEM of osteoblast-like cells in culture (DMEM supplemented with 2 g/ml of human fibronectin) on(A)non-sandblasted titaniumdisk surface (bar 10 m) and (B) on rough sandblasted Ti disk surface. Cells appear more flattened than with DMEM supplementewith 5% FCS (bar 10 m).

Table 3. Disintegrations per minute (dpm 103 SD) of tritiated thymidine incorporated intocells in culture on smooth (type I) and rough (type II) titanium disks and on control plasticsurface

Controls Type I Type II

24 hours (n 5) 277 32 (n 5) 266 13 (n 5) 290 15 NS

48 hours (n

5) 401 24

a

(n

5) 259 17

b

(n

5) 325 8

c

P 0.02 NS P 0.03

P 0.01a versus b/c; b versus c




strated that roughness modulates cytokine and growth factor(prostaglandin E2, TGF 1) production by MG63 osteo-blast-like cells [34].

In our study, the measurement of cell adhesion and pro-liferation with tritiated thymidine showed that proliferationof Saos-2 cells was decreased on both types of Ti diskscompared with plastic of the culture vials. In addition, theproliferation of cells in culture on smooth disks was signifi-cantly reduced than when cultured on rough disks. These

data agree with previous reports demonstrating that signifi-cantly higher levels of cellular attachment were found usingrough, sandblasted surfaces with irregular morphologies[30]. Our results may appear to conflict with a recent reportshowing that fibroblasts have a higher attachment to smooththan to rough Ti [35]. However, in this report, the morphol-ogy of the roughest Ti surfaces displayed a sharp shapingthat could damage or injure the cells. It appears that attach-ment and proliferation of cells depend not only on surfaceroughness but also on surface micromorphology (sharp orrounded edges) of the implant surface, a parameter thatappears difficult to quantify at the present time.

Attachment and spreading of cells on substrata involve

extracellular proteins adsorbed onto the surfaces, specificmembrane receptors, and cytoskeletal proteins. Serum pro-teins adsorb quickly after contact with an inorganic materialsurface such as Ti implants, therefore, the original surface isno longer in contact with the host tissue [36, 37]. Moreover,Ti surfaces with different oxide structures bind significantamounts of human serum albumin and fibronectin [38].

Fibronectin and vitronectin are major extracellular ma-trix proteins and are found in abundance in blood plasma[13, 39]. These proteins are known to be involved in celladhesion processes and contain the specific RGD sequencethat is recognized by specific cell membrane receptors suchas integrins. Integrins are transmembrane () het-erodimers, the intracellular part of which is linked to theactin microfilament network through talin and/or vinculin,tensin, and -actinin [4045]. Fibronectin and vitronectinare ligands for several integrins [13, 15, 41].

The binding of cells to extracellular proteins generatesan intracellular signal that affects the cytoskeleton status,initiates actin microfilament assembly and focal adhesionplates, and influences cell spreading [4547]. Because fi-bronectin and vitronectin were supposed to concern the celladhesion to the Ti surface, we have compared, in SEM,osteoblast-like cells cultured in the presence of FCS, orfibronectin or vitronectin.

In presence of FCS (containing adhesive proteins includ-ing fibronectin and vitronectin), cells showed a flattenedappearance with large spreadings and they covered a largepart of the culture area. In absence of FCS, the cells dis-played a globular shape and were packed in sparse clustersleaving most of the Ti surface uncovered. When fibronectinor vitronectin were added, cells were slightly more flattenedand spread than with FCS. However, fibronectin and vitro-nectin concentrations were not measured in the culture me-dium supplemented with 5% FCS. These results confirmthat fibronectin and vitronectin are involved in the initialphase of the cell adhesion processes and mainly influencecell attachment to the Ti surface and cell spreading [48, 49].Extracellular fibronectin induces rapid integrin receptor ag-

gregation and transmembrane accumulation of a variety ofcytoskeletal proteins including talin, -actinin, and tensin[44, 5053].

Several integrin subunits (e.g.,v

,1,

5,

6) have been

identified in bone cells and in the Saos-2 cell line [54].

Specific integrin subunits were demonstrated to binwell-defined ligands in the extracellular matrix. Fibronectiis the major ligand for

51, and the

v3

group of integrinbinds to vitronectin. Moreover, v3 was reported to bimplicated in the fibronectin matrix assembly [55]. In ouexperiment, the presence of anti-1integrin monoclonal antibody in the fibronectin-supplemented culture medium dramatically decreased cell adhesion, but did not affect adhesion and proliferation of cells cultured in the vitronectin

supplemented medium. Thus, the 1 integrin subunappears as the major ligand for fibronectin but not for vitronectin. The

v integrin subunit was identified to be in

volved in the binding to fibronectin and vitronectin [13]and in the assembly of fibronectin matrices [55]. This waconfirmed in our experiment showing that the cell morphology was altered with a globular shape when cultures werperformed in the presence of anti-vin the culture mediumsupplemented with vitronectin or fibronectin. The

vinte

grin subunit appears to be of major interest in the organization of cells that are bound to fibronectin or vitronectinIntegrin heterodimers involving vand 1are strongly implicated in attachment and/or spreading of Saos-2 osteo

blast-like cells to fibronectin or vitronectin-adsorbed substrata.The substratum surface topography alters cell shape an

modulates fibronectin at the transcriptional and posttranscriptional levels as well as the amount of fibronectin assembly into the extracellular matrix [56]. The surface texture of the Ti substrate can also affect the expression ofibronectin and vitronectin integrin receptors [57], modiftheir clustering or aggregation, and therefore determinvariations in shape and spreading of cells.

Acknowledgments. The authors would like to thank Pr. P. Allain

Department of Pharmacologie, CHU Angers, for ICPOES measurements, Dr. L. Fouilland, Ecole Nationale Superieure des Artet Metiers Angers, for profilometric determinations, and Euroteknika for preparing and supplying both types of titanium disks

References

1. Helsingen AL, Lyberg T (1995) Implants de Brnemark esystemes analogues. Implant 1:2332

2. Okabe T, Hero H (1995) The use of titanium in dentistry. CeMater 5:211230

3. Albrektsson T, Albrektsson B (1987) Osseointegration obone implants. a review of an alternative mode of fixation

Acta Orthop Scand 58:5675774. Boyan B, Hummert T, Dean D, Schwartz Z (1996) Role o

material surfaces in regulating bone and cartilage cell response. Biomaterials 17:137146

5. Buser D, Schenk RK, Steinemann S, Fiorellini JP, Fox CHStich H (1991) Influence of surface characteristics on bonintegration of titanium implants. A histomorphometric studin miniature pigs. J Biomed Mater Res 25:889902

6. Keller JC, Stanford CM, Wightman JP, Draughn RA, ZahariaR (1994) Characterizations of titanium implant surfaces. Biomed Mater Res 28:939946

7. Larsson C, Thomsen P, Aronsson BO, Rodahl M, Lausmaa JKasemo B, Ericson LE (1996) Bone response to surfacemodified titanium: studies on the early tissue response to ma

chined and electropolished implants with different oxidthicknesses. Biomaterials 17:6056168. Larsson C, Thomsen P, Lausmaa J, Rodahl M, Kasemo B

Ericson LE (1994) Bone response to surface-modified titanium: studies on electropolished implants with different oxidthicknesses and morphology. Biomaterials 15:10621074




9. Davies JE, Lowenberg B, Shiga A (1990) The bone-titaniuminterface in vitro. J Biomed Mater Res 24:12891306

10. Lowenberg B, Chernecky R, Shiga A, Davies JE (1991) Min-eralized matrix production by osteoblasts on solid titanium invitro. Cells Materials 1:177187

11. Clover J, Dodds RA, Gowen M (1992) Integrin subunit ex-pression by human osteoblasts and osteoclast in situ and inculture. J Cell Science 103:267271

12. Sinha RK, Morris F, Shah SA, Tuan RS (1994) Surface com-position of orthopdic implant metals regulates cell attach-

ment, spreading, and cytoskeletal organisation of primary hu-man osteoblasts in vitro. Clin Orthop Rel Res 305:25827213. Kreis T, Vale R (1993) Guidebook to the extracellular matrix

and adhesion proteins. Sambrook Tooze Publishers, OxfordUniversity Press, pp 5658

14. Puleo DA, Bizios R (1991) RGDS tetrapeptide binds to os-teoblasts and inhibits fibronectin-mediated adhesion. Bone 12:271276

15. Howlett CR, Evans MDM, Walsh WR, Johnson G, Steele JG(1994) Mechanism of initial attachment of cells derived fromhuman bone to commonly used prosthetic materials duringcell culture. Biomaterials 15:213222

16. Callen B, Lowenberg B, Lugowski S, Sodhi R, Davies J(1995) Nitric acid passivation of Ti6A14V reduces thicknessof surface oxide layer and increases trace element release. J

Biomed Mater Res 29:27929017. Lowenberg BF, Lugowski S, Chipman M, Davies JM (1994)

ASTM-F86 passivation increases trace elements release fromTi6Al4V into culture medium. J Mat Sci Mat Med 5:467472

18. Kawahara D, Ong JL, Raikar GN, Lucas LC, Lemons JE,Nakamura M (1996) Surface characterization of radio-frequency glow discharge and autoclaved titanium surfaces.Int J Oral Maxillofac Implants 11:435442

19. Wennerberg A (1996) On surface roughness and implant in-corporation. Thesis Goteborg University Sweden

20. Hure G, Donath K, Lesourd M, Chappard D, Basle MF (1996)Does titanium surface treatment influence the bone-implantinterface SEM and histomorphometry in a 6-month sheepstudy. Int J Oral Maxillofac Implants 11:506511

21. Lesourd M, Donath K, Chappard D, Basle MF (1996) Etat desurface et compatibilite in vivo de deux types dimplants entitane. Implant 2:5359

22. Martin J, Schwartz Z, Hummert T, Schraub D, Simpson J,Lankford J, Dean D, Cochran D, Boyan B (1995) Effect oftitanium surface roughness on proliferation, differentiationand protein synthesis of human osteoblast-like cells (MG63).J Biomed Mater Res 29:389401

23. Schwartz Z, Martin J, Dean D, Simpson J, Cochran D, BoyanB (1996) Effect of titanium roughness on chondrocytes pro-liferation, matrix production and differentiation depends onthe state of cell maturation. J Biomed Mater Res 30:145155

24. Bianco P, Ducheyne P, Cuckler J (1996) Local accumulationof titanium released from a titanium implant in absence ofwear. J Biomed Mater Res 31:227234

25. Bianco P, Ducheyne P, Cuckler J (1996) Titanium serum andurine levels in rabbits with a titanium implant in the absenceof wear. Biomaterials 17:19371942

26. Wang J, Tsukayama D, Wicklund B, Gustilo R (1996) Inhi-bition of T and B cell proliferation by titanium, cobalt, andchromium: role of IL-2 and IL-6. J Biomed Mater Res 32:655661

27. Haynes D, Rogers S, Hay S, Pearcy M, Howie D (1993) Thedifferences in toxicity and release of bone-resorbing mediatorsinduced by titanium and cobalt-chromium-alloy wear par-ticles. J Bone Joint Surg 75-A:825834

28. Wang J, Wicklund B, Gustilo R, Tsukayama D (1996) Tita-nium, chromium and cobalt ions modulate the release of bone-associated cytokines by human monocytes/macrophages in

vitro. Biomaterials 17:2233224029. Elagi K, Vernon C, Hildebrand H (1995) Titanium-inducedenzyme activation on murine peritoneal macrophages in pri-mary culture. Biomaterials 16:13451351

30. Bowers KT, Keller JC, Randolph BA, Wick DG, MichaelsCM (1992) Optimization of surface micromorphology for en-

hanced osteoblast responses in vitro. Int J Oral MaxillofaImplants 7:302309

31. Goldberg V, Stevenson S, Feighan J, Davy D (1995) Biologof grit-blasted titanium alloy implants. Clin Orthop 319:122129

32. Ungersbock A, Pohler O, Perren S (1996) Evaluation of sotissue reactions at the interface of titanium limited contacdynamic compression plate implants with different surfactreatments: an experimental sheep study. Biomaterials 17797806

33. Wennerberg A, Albrektsson T, Anderson T (1996) Bone tissuresponse to commercially pure titanium implants blasted witfine and coarse particles of aluminium oxide. Int J Oral Maxillofac Implants 11:3845

34. Kieswetter K, Schwarz Z, Hummert T, Cochran D, Simpson Dean D, Boyan B (1996) Surface roughness modulates thlocal production of growth factors and cytokines by osteoblast-like MG-63 cells. J Biomed Mater Res 32:5563

35. Cochran D, Simpson J, Weber H, Buser D (1994) Attachmenand growth of periodontal cells on smooth and rough titaniumInt J Oral Maxillofac Implants 9:289297

36. Olivieri M, Rittle K, Tweden K, Loomis R (1992) Comparative biophysical study of adsorbed calf serum, fetal bovinserum and mussel adhesive protein films. Biomaterials 13

20120837. Kothari S, Hatton P, Douglas C (1995) Protein adsorption t

titania surfaces. J Mater Sci-Mater Med 6:69569838. Walivaara B, Aronsson B, Rodahl M, Lausmaa J, Tengvall

(1993) Titanium with different oxides: in vitro studies of protein adsorption and contact activation. Biomaterials 15:827834

39. Robey PG, Boskey AL (1996) The biochemistry of boneOsteoporosis. Marcus R, Feldman D, Kelsey J (eds) AcademiPress, New York, pp 95183

40. Brighton C, Albelda S (1992) Identification of integrin celsubstratum adhesion receptors on cultured rat bone cells. Orthopaed Res 10:766773

41. Diamonds M, Springer T (1994) The dynamic regulation o

integrin adhesiveness. Curr Biol 4:50651742. Grzesik WJ, Gehron Robey P (1994) Bone matrix RGD gly

coproteins: immunolocalization and interaction with humaprimary osteoblastic bone cells in vitro. J Bone Miner Re9:487496

43. Gronowicz G, McCarthy M (1996) Response of human osteoblasts to implant materials: integrin-mediated adhesion. J Orthopaed Res 14:878887

44. Yamada K, Miyamoto S (1995) Integrin transmembrane signaling and cytosol control. Curr Biol 7:681689

45. Meredith J, Winitz S, Lewis M, Hess S, Ren X, Renshaw MSchwarz M (1996) The regulation of growth and intracellulasignaling by integrins. Endocr Rev 17:207220

46. Oakley C, Brunette D (1993) The sequence of alignment omicrotubules, focal contacts and actin filaments in fibroblastspreading on smooth and grooved titanium substrata. J CeSci 106:343354

47. Garrat A, Humphries M (1995) Recent insights into liganbinding, activation and signaling by integrin adhesion receptors. Acta Anat 154:3454

48. Dean J, Culbertson K, DAngelo A (1995) Fibronectin anlaminin enhance gingival cell attachment on dental implansurfaces in vitro. Int J Oral Maxillofac Implants 10:72172

49. Eisenbarth E, Meyle J, Nachtigall W, Breme J (1996) Influence of the surface structure of titanium materials on the adhesion of fibroblasts. Biomaterials 17:13991403

50. Plopper G, Ingber D (1993) Rapid induction and isolation ofocal adhesion complexes. Biochem Biophys Res Commu193:571578

51. Miyamoto S, Akiyama S, Yamada K (1995) Synergistic rolefor receptor occupancy and aggregation in integrin transmembrane function. Science 267:883885

52. Miyamoto S, Teramoto H, Coso O, Gutkind J, Burbelo P




Akiyama S, Yamada K (1995) Integrin function: molecularhierarchies of cytoskeletal and signaling molecules. J CellBiol 131:791805

53. Plopper G, McNamee H, Dike L, Bojanowski K, Ingber D(1995) Integration of integrin and growth factor receptor sig-naling pathways within the focal adhesion complex. Mol BiolCell 6:13491365

54. Chiang HS, Yang RS, Huang TF (1995) The Arg-Gly-Asp-containing peptide, rhoostomin, inhibits in vitro cell adhesionto extracellular matrices and platelet aggregation caused by

Saos-2 human osteosarcoma cells. Brit J Cancer 71:265266

55. Wu C, Hughes PE, Ginsberg MH, McDonald JA (1996) Identification of a new biological function for integrin V-3initiation of fibronectin matrix assembly. Cell Adhes Commun 4:149158

56. Chou L, Firth J, Uitto V, Brunette D (1995) Substratum surface topography alters cell shape and regulates fibronectimRNA level, mRNA stability, secretion and assembly in human fibroblasts. J Cell Sci 108:15631573

57. Hormia M, Kononen M (1994) Immunolocalization of fibronectin and vitronectin receptors in human gingival fibroblast

spreading on titanium surfaces. J Periodontal Res 29:14615


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