Attachment, proliferation and osteogenic response of osteoblast-like cells cultured on titanium...

Attachment, Proliferation and Osteogenic Response ofOsteoblast-Like Cells Cultured on Titanium Treatedby a Novel Multiphase Anodic Spark Deposition Process

Elena De Angelis,1 Francesca Ravanetti,2 Antonio Cacchioli,2 Attilio Corradi,1 Carmen Giordano,3

Gabriele Candiani,3 Roberto Chiesa,3 Carlo Gabbi,2 Paolo Borghetti1

1 Pathology Unit, Department of Animal Health, Faculty of Veterinary Medicine, University of Parma, Parma, Italy

2 Anatomy Unit, Department of Animal Health, Faculty of Veterinary Medicine, University of Parma, Parma, Italy

3 Department of Chemistry, Materials and Chemical Engineering, Politecnico di Milano, Milan, Italy

Received 21 May 2007; revised 19 March 2008; accepted 9 April 2008Published online 7 August 2008 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.b.31179

Abstract: A new bioactive titanium surface treatment, labeled Ti-ASD, was developed using

the electrochemical anodic spark deposition (ASD) technique and results in a thickened

titanium oxide layer with higher levels of calcium and phosphorus typical of newly deposited

mineral phase. This study was aimed at extending the knowledge on Ti-ASD treatment, by

means of evaluation of the attachment, morphology, proliferation, metabolic activity,

differentiation, and mineralization of osteoblast-like cells (SaOS-2) after growth on Ti-ASD

treated titanium compared with nontreated titanium (Ti) and with chemically etched titanium

(Ti-ETC). This novel type of titanium coating supported cell attachment, cell proliferation,

and mineralization, revealing no cytotoxicity effects. The expression of differentiation markers

on Ti-ASD treated titanium shows that genes related to the proliferation phase (Collagen type

I, Coll I; Cbfa-1) were early expressed, whereas genes related to the mineralization phase

(alkaline phosphatase, osteopontin, bone sialo protein) increased in a time-related way.

Mineralization occurred on all analyzed surfaces, but on Ti-ASD the number of bone-like

nodules and the amount of mineralized area was higher. In conclusion, Ti-ASD resulted to be a

good surface for osteoblast attachment and proliferation, also promoting the maintenance of

cell differentiation and matrix mineralization, a fundamental requirement for sustain the

osseointegration and the clinical success of dental implants. ' 2008 Wiley Periodicals, Inc. J Biomed

Mater Res Part B: Appl Biomater 88B: 280–289, 2009

Keywords: titanium; surface treatment; human osteoblast-like cells; attachment; differ-

entiation

INTRODUCTION

For clinically successful implants, interaction between

implanted materials and surrounding tissue is critical.

Osseointegration is the condition in which mature bone is

deposited on implant without any interposed soft or fibrous

tissue. For dental and orthopedic application, a basic require-

ment for osseointegration is the type of interaction between

implant surface and the surrounding tissue. The bone pro-

vides a dynamic interface in continuous evolution1,2 and

then the osseointegration process is complex and involves

numerous factors in which a key role is played by topogra-

phy and chemistry of the implant surface3; a suitable surface

for osseointegration should first promote cell attachment,

and then sustain cell proliferation, maintain cell differentia-

tion, and improve extracellular matrix secretion.2 While dif-

ferentiation into bone-forming cells is the final parameter for

a good material–cell contact, the first period of implantation

is very important for cell attachment to the surface. In vitromodels, using cell culture, provide useful information relat-

ing to cell–biomaterial interaction.4,5 Cell culture parameters

(cell attachment, cell proliferation, differentiation markers,

matrix mineralization) can be used to screen in vitro syn-

thetic biomaterials prior to in vivo testing.

One of the main aims of the materials research for

implantology is related to the study and development of

surface modification techniques to improve the bone inte-

gration of titanium-based dental implants. Several mechani-

Correspondence to: E. De Angelis (e-mail: [email protected])Contract grant sponsor: MIUR (COFIN 2002 and COFIN 2004)

' 2008 Wiley Periodicals, Inc.

280

cal, chemical, and electrochemical techniques are currently

used to improve the surface of dental implants: acid etch-

ing, electropolishing, electrochemical treatments such as

anodic oxidation, sand blasting, and plasma spraying.6,7

A new bioactive titanium surface treatment, labeled Ti-

ASD, was developed using the electrochemical anodic

spark deposition (ASD) technique8,9: Ti-ASD treatment

involves the sequence of two ASD treatments followed by

a final alkali etching. The treatment is capable to properly

modify the superficial titanium oxide film. Ti-ASD treat-

ment results in a thickened titanium oxide layer with higher

levels of calcium and phosphorus. This layer was proven to

show excellent mechanic stability, high mineralization

potential, preferential protein adsorption, and high potenti-

ality in stimulating osteoblast activity.10,11

This study was aimed at extending the knowledge on

Ti-ASD treatment, by means of evaluation of the attach-

ment, morphology, metabolic activity, proliferation, differ-

entiation, and mineralization of a osteoblast cell line

(SaOS-2)12 after growth on Ti-ASD treated titanium com-

pared with non-treated titanium, labeled Ti, and with

chemically etched titanium, labeled Ti-ETC. Osteoblast dif-

ferentiation was analyzed by gene expression of specific

markers of osteoblast phenotype: collagen type I (Coll I)

which is associated with formation of the extracellular ma-

trix in proliferative step, alkaline phosphatase (ALP) which

is expressed during the first phase of the matrix maturation

step, osteopontin (OPN) which is expressed during the ma-

trix maturation step, bone sialo proteins (BSPs) and osteo-

calcin (OC) which are expressed in the mineralization

step.13,14 A key regulator of osteoblast differentiation is the

transcription factor Runx2 (core-binding factor 1, Cbfa1)15;

Runx2 binds to the osteoblast-specific cis-acting element 2

(OSE2), found in the promoter regions of all the major

osteoblast-specific genes (ALP, OPN, OC), and controls

their expression.16

MATERIALS AND METHODS

Preparation of Samples

All titanium specimens (3.0 cm 3 2.5 cm 3 1 mm) were

obtained from a commercially pure, grade 2 titanium sheet

(Torresin Titanio Metalli S.r.l., Padova, Italy). Three differ-

ent titanium surface treatments were investigated in this

study: (a) untreated, machined titanium, Ti, was used as a

control material; (b) chemically treated titanium, Ti-ETC,

resulting from a decontamination by strong alkali etching

followed by a double-step acid etching: the first step

carried out in 1M NaOH containing 2% H2O2 at 808C for

10 min, the second one in an acid water solution at 288Cfor 1 h; (c) electrochemically treated titanium, Ti-ASD,10

was prepared in an electrochemical cell through two con-

secutive ASD treatments, the first one performed in a solu-

tion containing calcium and phosphate ions and the second

one in a solution containing calcium ions alone. A final

treatment in concentrated potassium hydroxide water solu-

tion at 608C was then performed. Materials were treated

and supplied by the Department of Chemistry, Materials

and Chemical Engineering ‘‘G.Natta,’’ Politecnico di Mi-

lano, Milano, Italy. Before use, materials were sterilized by

rinsing them twice in pure ethanol for 30 s, washed in ster-

ile bidistilled H2O, and then dried in a laminar flow hood.

Cell Culture

Human SaOS-2 cells (provided by Cell Culture Laboratory,

Istituto Zooprofilattico Sperimentale della Lombardia e del-

l’Emilia Romagana ‘‘Bruno Ubertini,’’ Brescia, Italy; Experi-

mental Institute of Zooprophylaxis of Brescia, Italy) were

cultured in McCoy’s 5A medium containing 15% fetal calf

serum (FCS), 100 U/mL penicillin, and 0.1 mg/mL strepto-

mycin. Cells were seeded on the three different titanium sur-

faces at the density of 2 3 104 cells/cm2. Incubation was

carried out at 378C in a humidified atmosphere of 5% CO2

in air for the time specified for each single experiment.

Scanning Electron Microscopy

Scanning electron microscopy (SEM) observation was per-

formed after 6, 24, 48 h, and 4 days of culture. Culture for

SEM analysis was performed in triplicate. Cells grown on

different titanium surfaces were fixed at each selected time

point with 2.5% glutaraldehyde in 0.1M sodium cacodylate

buffer (pH 5 7.3) for 2 h at 48C. They were then dehy-

drated through a series of alcohols and then critical point

dried with liquid carbon dioxide (CPD 030 Baltec, Wallruf,

Germany). Specimens were then sputter-coated (Balzers de-

vice) with gold-palladium (Plano, Germany) using a SCD

040 coating device (Balzer Union, Wallruf, Germany).

Samples were observed using a Zeiss DSM 950 scanning

electron microscope at an accelerating voltage of 10 kV

(Zeiss, Jena, Germany).

Cell Attachment, Cell Proliferation, and Cell Viability

For the evaluation of cell proliferation, cells were cultured

on materials for 1, 2, 4, and 7 days. The 1-day time-point

was used to evaluate cell attachment. At each time point, af-

ter medium removing, surfaces were washed with PBS and

then incubated with trypsin (0.1% trypsin – 0.02% EDTA

(Sigma-Aldrich, St. Louis, MO) for 7 min to obtain cell

detachment. Obtained cells were counted by Burker’s hemo-

cytometer. For cell viability evaluation, detached cells were

incubated for 5 min with Trypan blue and then counted.

Complete removal of the attached cells from the surfaces

with trypsin-EDTA was confirmed by SEM. Values reported

in the graph represent the mean 6 standard deviation of

three independent analyses performed in triplicates.

281GROWTH OF OSTEOBLAST-LIKE CELLS ON ASD-TREATED TITANIUM

Journal of Biomedical Materials Research Part B: Applied Biomaterials

MTT Assay

Cell viability was also evaluated by measuring the mito-

chondrial dehydrogenase activity using a modified MTT (3-

(4,5-dimetyl-2-tiazolyl)-2,5-diphenyl-2H-tetrazolium bro-

mide) reduction assay. Briefly, the cells on days 1, 2, 4,

and 7 after seeding on the titanium surfaces were incubated

with 600 lL McCoy’s 5A and 60 lL MTT (Sigma-Aldrich;

Cat. No. M2128), 5 mg/mL in PBS for 4 h at 378C. Then,600 lL of 10% SDS in 0.01M HCl was added and incu-

bated overnight at 378C. The resulting solution was moved

into fresh plates and the absorbance was measured using

Spectra Shell Microplate Reader (SLT, TECAN, Milan,

Italy) at a wavelength of 540 nm (the reference value was

690 nm). Three titanium surfaces without cells incubated

with medium and MTT was used as control samples

(blank).

mRNA Isolation and RT-PCR

Total RNA was extracted with a RNAwiz kit (Ambion,

Austin, TX), according to manufacturer’s instruction, from

cell samples after 4 and 7 days of culturing and then spec-

trophotometrically quantified. Two micrograms of total

RNA was used for reverse-transcribing to cDNA using

‘‘Ready-To-Go You-Prime First-Strand Beads’’ (GE Health-

care, Uppsala, Sweden) in a reaction volume of 33 lL.Obtained cDNA was used as a template for polymerase

chain reaction with specific primers for Cbfa1, type I colla-

gen, ALP, OPN, and BSPs. Glyceraldehyde-3-phosphate

dehydrogenase (GAPDH) was used as housekeeping gene

(Table I). Five microliters of cDNA was amplified using

DyNAZyme II DNA polymerase Recombinant (Ambion),

and 5 lL of both forward and reverse primers (25 lM).

The PCR products were separated by electrophoresis in a

2% agarose gel in Tris-acetate-EDTA buffer and visualized

by ethidium bromide staining under UV light. The density

of each band was quantified by densitometric analysis with

Scion Image (Scion Capture Driver 1.2 for Image-Pro Plus;

Scion, MA).

Mineralization Staining (Alizarin Red S)

Human SaOS-2 cells were plated in triplicate at the density

of 2 3 104 cell/cm2 onto prepared titanium samples. Cells

were cultured in osteogenic culture medium, which consists

of McCoy’s 5A medium, 15% FCS, 100 U/mL penicillin,

0.1 mg/mL streptomycin, 250 lM L-ascorbic acid, 10 mMb-glycerophosphate, and 10 nM of dexamethasone. Also,

control samples (each biomaterial without cells and with

osteogenic medium) were run. After 21 days of culture,

Alizarin Red staining was performed. Cells were washed

twice with PBS and fixed in 4% paraformaldehyde in 0.1MPBS for 20 min at room temperature and then rinsed once

with deionized water. After fixation, the cultures were

stained with 2% solution of Alizarin Red S in 0.1M PBS

for 5 min at room temperature. Excess dye was washed off

with deionized water. Samples were visualized through a

microscopy (Nikon SMZ1000; Nikon, Tokyo, Japan). The

titanium area covered with a red stain, representing miner-

alized bone-like nodules, was then measured with an image

analysis system (Laboratory Universal Computer Image

Analysis-LUCIA G, Version 5.0, Laboratory Imaging,

Prague, Hungary). For the evaluation of mineralization pat-

tern, the number of nodules, the area of titanium surface

covered by each single bone nodule (bone-nodule area),

and the total area of titanium surface covered by the bone

nodules (total bone-nodules area) were measured.

Statistical Analysis

Using a statistical software program (SPSS, Chicago, IL)

for the variance test (ANOVA) and Tukey’s multiple com-

parison test post hoc, data were analyzed to highlight any

significant differences between surface treatments. Statisti-

cally significant differences (p \ 0.05 or p \ 0.001) were

indicated in the text, figures, and table.

TABLE 1. Oligonucleotide Primers for Gene Expression Analysis by RT-PCR

Gene Primer Sequence Tm (8C)

Expected

Product

Size (bp)

ALP Sense: 50 GCG AAA GTA TTT CTC CAG ACC CAG 30 64.4 367

Antisense: 50 TTC CAA ACA GGA GAG TCG CTT CAA 30

OPN Sense: 50 TGA GAG CAA TGA GCA TTC CGT TG 30 60.6 330

Antisense: 50 CAG GGA GGT TCC ATG AAG CAA C 30

Cbfa1 Sense: 50 CTC TTC CCA AAG CCA GAG TG 30 59.4 205

Antisense: 50 CAG CGT CAA CAC CAT CAT TC 30

Type I Collagen Sense: 50 CCT CAA GGG CTC CAA CGA G 30 57.3 117

Antisense: 50 TCA ATC ACT GTC TTG CCC CA 30

BSP Sense: 50 ATT GAA AAC GAA AGC GAA G 30 51.5 450

Antisense: 50 ATC ATA GCC ATC GTA GCC TTG T 30

GAPDH Sense: 50 GAA GGT GAA GGT CGG AGT C 30 45 224

Antisense: 50 GAA GAT GGT GAT GGG ATT TC 30

282 DE ANGELIS ET AL.


RESULTS

Scanning Electron Microscopy

All titanium surface treatments supported cell adhesion and

proliferation; however, this process showed different dy-

namics related with the peculiar surface topography of each

material. Cells began to adhere and spread on the materi-

als’ surfaces within a short time after being seeded. After 6

h (Figures 1 and 2), SaOS-2 cells were adherent with an

heterogeneous morphology: some cells were elongated,

other polygonal or rounded. Cells mostly presented a typi-

cal morphology of osteoblast, a central spherical body with

the cytoplasm extending away from the central area in all

directions and adhering to the titanium surfaces with fila-

mentous protrusions.

After 4 days on Ti and Ti-ETC, cells covered the mate-

rial uniformly with an almost confluent monolayer. Particu-

larly, on Ti-ETC, (Figure 2) the cells prevalently had a

polygonal shape, with many adhesion points at the surface,

creating a spreading of cells and presenting a more flat-

tened morphology than that of cells growing on Ti. After 4

days of culturing, cells were tightly flattened and single

cells were difficult to distinguish from each other.

After 24 h on Ti-ASD surface (Figure 3), cells were still

relatively spread on the surface, and presented a rounded

morphology with many cytoplasmic processes extended

from cytoplasm to material surface. After 48 h, cells had

increased the extent of cell spreading and appeared homo-

geneously attached on Ti-ASD but still inconfluent and

with few intercellular contacts. After 4 days, cells were

extensively spread, exhibiting a highly flattened morphol-

ogy. Cells were frequently attached to each other with

scarce intercellular gaps, forming a regular monolayer. At

each time point cells grown on Ti-ASD presented many ap-

ical ruffles, testifying a high cellular activity.17 On Ti and

Ti-ETC this aspect was less evident and it appeared only

within 48 h after seeding.

Cell Attachment and Cell Proliferation

Cell attachment, evaluated 1 day after seeding, turned out

to be similar on Ti and Ti-ETC, while the number of cells

Figure 1. SEM morphology (magnification 3640) of SaOS-2 cells after 6, 24, 48 h, and 4 days of

growing on titanium Ti.



attached on Ti-ASD was statistically significantly lower

(Ti-ASD vs Ti and Ti-ETC, p \ 0.05) as shown in Figure

4. On all titanium surfaces the cell proliferation rate

increases in a time-related way with similar kinetics

(Figure 4); however, after 2, 4, and 7 days of culture, cell

numbers resulted higher on Ti and Ti-ETC than on Ti-

ASD. Particularly, at 2 days Ti-ETC compared with Ti-

ASD results statistically significant (p\ 0.05), at 4 days Ti

compared with Ti-ASD results statistically significant (p 50.05), and at 7 days Ti and Ti-ETC compared with Ti-ASD

results statistically significant (Ti-ASD vs Ti and Ti-ETC

p \ 0.05). Cell viability was evaluated by Trypan Blue

assay; results were similar at each time point on Ti, Ti-

ETC, and Ti-ASD: at least 95% of recovered osteoblasts

was viable (data not shown).

MTT Assay

The metabolic activity, measured by MTT assay, was nor-

malized for the cell numbers counted on the surfaces at each

time point (Figure 5). At all time points the metabolic activ-

ity of cells resulted higher on Ti-ASD when compared with

Ti-ETC and Ti (at 1 day: Ti-ASD vs Ti and Ti-ETC, p \0.05; at 2, 4, and 7 days: Ti-ASD vs Ti and Ti-ETC, p \0.001). With time, the metabolic activity of cells on Ti and

TI-ETC presented a similar trend characterized by an

increase at 2 days, but statistically significant for Ti-ETC (1

vs 2 days, p \ 0.001) because only for Ti-ETC it kept high

up to 4 days (1 vs 4 days, p \ 0.001), with a subsequent

decrease at 4 days for Ti (2 days vs 4 and 7 days, p \0.001) and a slightly delayed decrease for Ti-ETC (7 days vs

2 and 4 days, p \ 0.001). Only at 4 days there was differ-

ence between Ti and Ti-ETC (Ti vs Ti-ETC, p \ 0.001). At

2 days, on Ti-ASD the metabolic activity statistically signifi-

cantly increased, and then beginning to slowly decrease (1

day vs 2 and 4 days, p \ 0.001; 2 days vs 4 and 7 days,

p\ 0.001; 4 days vs 7days, p\ 0.001); at 4 days the cellu-

lar metabolic activity was lower when compared with the

value reached at 2 days, but still high, and finally at 7 days it

turned out comparable with the 1-day time-point.

Figure 2. SEM morphology (magnification 3640) of SaOS-2 cells after 6, 24, 48 h, and 4 days of

growing on titanium Ti-ETC.



RT-PCR

The analysis and quantification of mRNA expression for

specific osteoblast markers was carried out by reverse tran-

scription PCR after 4 and 7 days of SaOS-2 cells culture

on Ti, Ti-ETC, and Ti-ASD. Specific osteoblast markers

Cbfa-1, ALP, OPN, BSP, and type I collagen were used.

Data showed that Cbfa-1, type I collagen, ALP, OPN,

and BSP were expressed in all samples at each experimen-

tal time although there are some differences in the genes’

expression (Figure 6) as pointed out by densitometric anal-

ysis (Figure 7). The Cbfa-1 expression resulted similar on

the different surfaces (Ti, Ti-ETC, Ti-ASD) at each time

point. The ALP was expressed in all samples at each time:

after 4 days of culture ALP expression ended up being sim-

ilar for cells growing on Ti and Ti-ETC, whereas it was

lower for cells growing on Ti-ASD (Ti-ASD vs Ti and Ti-

ETC, p \ 0.001). Nevertheless, after 7 days of culture,

ALP expression on Ti-ASD increased (Ti-ASD: 4 days vs

7 days, p \ 0.001) and turned out to be similar among the

materials Ti, Ti-ETC, and Ti-ASD. The analysis of type I

collagen expression on testing materials showed no striking

differences at each experimental time; however, after 4

days of culture on Ti and Ti-ETC, type I collagen expres-

sion turned out to be lower compared with Ti-ASD (Ti-

ASD vs Ti and Ti-ETC, p \ 0.001), whereas at 7 days Ti

and Ti-ETC reached the Ti-ASD value (Ti: 4 days vs 7

days, p \ 0.001; Ti-ETC: 4 days vs 7 days, p \ 0.001).

After 4 days of culture, OPN expression resulted to be

lower on Ti and Ti-ASD compared with Ti-ETC (Ti-ETC

vs Ti and Ti-ASD, p\ 0.001), but after 7 days it had risen

to the level recorded on Ti-ETC (Ti: 4 days vs 7 days, p\0.001; Ti-ASD: 4 days vs 7 days, p \ 0.001), which

showed no change. The expression of BSPs mRNA was

higher on Ti-ASD than on Ti and Ti-ETC after 4 days (Ti-

ASD vs Ti and Ti-ETC p \ 0.001), although differences

among the tested materials were not detectable after 7 days

because the BSP expression on Ti and Ti-ETC had risen to

the level recorded on Ti-ASD (Ti: 4 days vs 7 days, p \0.001; Ti-ETC: 4 days vs 7 days, p\ 0.001).

Mineralization Staining

Mineralization of extracellular matrix was evaluated after

Alizarin Red staining and quantified by the image analysis

Figure 3. SEM morphology (magnification 3640) of SaOS-2 cells after 6, 24, 48 h, and 4 days

growing on titanium Ti-ASD.



program (LUCIA G). The number of nodules, the area of

titanium surface covered by a bone nodule, and the total

area of titanium surface covered by the bone nodules were

measured (Table II). Bone-like nodules formation occurred

on all titanium surfaces but not on the negative control

samples (Figure 8). On Ti-ASD surface, the number of the

nodules was significantly higher when compared with Ti

(p \ 0.001) and Ti-ETC (p \ 0.001). The bone nodule

area on Ti-ASD and Ti-ETC surfaces resulted statistically

significantly lower when compared with Ti (Ti vs Ti-ASD,

p \ 0.001; Ti vs Ti-ETC, p \ 0.001). On Ti-ASD, an

increase in the amount of total nodules area was detected,

but it was not statistically significant when compared with

Ti and Ti-ETC. In conclusion, the mineralization pattern

had different dynamics on the three tested surfaces; particu-

larly, on Ti surface few and big nodules were present,

whereas on Ti-ETC and Ti-ASD surfaces numerous smaller

nodules were detectable.

DISCUSSION

In this study, osteoblast-like cell adaptation on three differ-

ent titanium surface treatments (Ti, Ti-ETC, Ti-ASD) was

tested. Cellular attachment, metabolic activity, proliferation,

differentiation, and mineralization were analyzed as param-

eters to characterize cell–biomaterial interaction.

SaOS-2 cells and other osteoblast-like cell lines are usu-

ally used to evaluate potential biomaterials in in vitro stud-

ies. This cell line expresses genes related to the

osteogenesis process, representing a useful model for the

evaluation of differentiation, matrix production, and miner-

alization. This provides useful information relating to cell–

biomaterial interaction and bone formation on the implant

surface.

In this study, cell attachment results to be similar on Ti

and Ti-ETC, whereas the number of cells attached on Ti-

ASD after 24 h was lower. The growth curves did not

achieve a plateau phase indicating no cytotoxic effects but

a good proliferation rate, and suggested similar kinetics on

the different titanium surfaces. However, cell proliferation

analysis showed that on Ti and Ti-ETC samples cell

growth was higher compared with Ti-ASD samples. These

Figure 6. Figure shows Cbfa-1, Collagen type I, ALP, OPN, BSP,

and GAPDH expression of the cells grown on Ti, Ti-ETC, Ti-ASD af-ter 4 and 7 days analyzed by RT-PCR (representative case).

Figure 4. Cell proliferation expressed as cell number 3 104/sample

(7.5 cm2) at 1, 2, 4, and 7 days of culture. Data are reported asmean 6 standard deviation (n 5 3). Tukey’s multiple comparison

test post hoc between tested surfaces for each experimental time:

*At day 1 Ti-ASD versus Ti and Ti-ETC (p\ 0.05); **at day 2 Ti-ASDversus Ti-ETC (p\ 0.05); 8at day 4 Ti versus Ti-ASD (p 5 0.05); 88atday 7 Ti-ASD versus Ti and Ti-ETC (p\ 0.05).

Figure 5. MTT assay expressed as optical density at 1, 2, 4, and 7

days of culture normalized for cell number counted on materials

at each time point. Data are reported as mean 6 standard deviation

(n 5 3). Tukey’s multiple comparison test post hoc between testedsurfaces for each experimental time: *At day 1 Ti-ASD versus Ti and

Ti-ETC (p \ 0.05); **at days 2, 4, and 7 Ti-ASD versus Ti and Ti-

ETC (p\ 0.001). Tukey’s multiple comparison test post hoc betweenexperimental times for each tested surface: Ti: a2 days versus 4 and

7 days (p\ 0.001); Ti-ETC: b1 day versus 2 and 4 days (p\ 0.001);c7 days versus 2 and 4 days (p \ 0.001); Ti-ASD: d1 day versus 2

and 4 days (p \ 0.001); e2 days versus 4 and 7 days (p\ 0.001); f4days versus 7 days (p\ 0.001).



data are in contrast with those of Giordano et al.,18 which

indicated a more flattened cellular morphology with cell

filopodia and a much higher activity for the cells cultured

on Ti-ASD, suggesting a more advanced attachment state

and proliferation activity on Ti-ASD surface. The described

discrepancy could be due to the different cell line used: it

is well known in literature that different cell lines respond

in different ways to the same surface features.19

Figure 7. Densitometric analysis of PCR product normalized with the GAPDH corresponding value.

Results are expressed as relative arbitrary units (RAU). Tukey’s multiple comparison test post

hoc between tested surfaces for each experimental time: *At day 4 Ti-ASD versus Ti and Ti-ETC

(p \ 0.001); **at day 4 Ti-ETC versus Ti and Ti-ASD (p \ 0.001). Tukey’s multiple comparison testpost hoc between experimental times for each tested surface: Ti: a4 days versus 7 days (p \ 0.001);

Ti-ETC: b4 days versus 7 days (p\ 0.001); Ti-ASD: c4 days versus 7 days (p\ 0.001).

TABLE 2. Data Regarding Bone-Like Nodules Formation in Cell Culture onto Ti, Ti-ETC, and Ti-ASD

Ti Ti-ETC Ti-ASD

Number of nodules/cm2 9.00 6 2.00 24.67 6 3.51 51.67 6 5.51*

Bone-nodule area (lm2) 30,895.24 6 782.13** 9503.26 6 5441.00 7011.02 6 4085.89

Total bone-nodules area (lm2) 277,139.28 6 50,966.26 232,612.80 6 30,215.32 364,981.02 6 42,572.53

Values represent the mean 6 standard deviation of triplicate analyses. Tukey0s multiple comparison test post hoc between tested surfaces: *Ti-ASD versus Ti and Ti-ETC

(p\ 0.001); ** Ti versus Ti-ETC and Ti-ASD (p\ 0.001).



The MTT analysis confirms that no cytotoxicity effect

was present in cells grown on Ti- ASD compared with Ti

and Ti-ETC. Cells grown on Ti-ASD demonstrated not

only a good vitality but, moreover, a higher metabolic ac-

tivity. The differences in the cell number among Ti-ASD

and the other two tested titanium surfaces is not caused by

cell death on Ti-ASD but by a lower proliferation on this

surface. The higher metabolic activity observed on this ma-

terial was probably due to a major matrix proteins produc-

tion (Collagen Type I, BSPs).

Several studies suggested that materials being able to pro-

mote an early cell attachment and a good proliferation rate

are not necessarily the best substrates on which differentia-

tion can occur at all.20,21 Meyer et al.21 demonstrated that

the material property being responsible for the different rates

of attachment is not the same as that influencing differentia-

tion on the material. Consequently, to test new biomaterials

it is important to study both cell attachment to biomaterials

and proliferation kinetics in relation to their subsequent dif-

ferentiation. The osteoblasts developmental sequence, associ-

ated to bone cell differentiation, started with a period of

active proliferation but showed a slow down in the cell divi-

sion rate accompanying an increase in the amount of matrix

production (a signal of differentiation).22 For the evaluation

of these aspects RT-PCR analysis of genes related to the

osteogenesis process was carried out.

Cbfa-1 is a transcription factor of genes related to the

osteogenesis process23; its expression turned out to be simi-

lar on the different surfaces tested in this study, thus sug-

gesting that osteogenic efficiency of cells was maintained

in each condition. Type I collagen, an essential matrix pro-

tein, plays a fundamental role in the maintenance of osteo-

blastic phenotype making the matrix competent for

mineralization. Results showed a similar expression on

each tested material testifying a specific extracellular ma-

trix production. The ALP is expressed during the osteoblast

proliferation phase and it reaches the highest enzymatic ac-

tivity in the mineralization process, where it plays a key

role. After 4 days of culture, ALP expression was slightly

lower for cells growing on Ti-ASD compared with the

other materials; however, after 7 days of culture, it ended

up being similar between Ti-ASD and the other materials.

OPN is a matrix protein with different functions. It plays a

primary role in regulating mineralized bone formation and

remodeling.24 OPN synthesis is related with ALP activity,

because ALP removes a phosphate group from glycerol

phosphate which enters into the cell and upregulates OPN

mRNA expression.25 In accordance with this evidence, our

results indicate that OPN expression is comparable with

ALP expression. Particularly, after 4 days of culture, OPN

expression resulted to be slightly higher for cells growing

on Ti-ETC compared with the other materials. After 4 days

of culture the BSP expression was slightly higher for cells

growing on Ti-ASD compared with other materials. How-

ever, after 7 days of culture, it ended up reaching similar

levels in BS and in the other materials. These results sug-

gest a more differentiated state of SaOS-2 cells grown on

Ti-ASD in comparison with cells grown on the other

materials; in fact, its expression is tightly associated

to bone mineralization participating in the nucleation of

hydroxyapatite.26

Obtained results show that the gene expression of Cbfa-

1, ALP, OPN, BSP, and type I collagen were positively

regulated by titanium surface treatments. Particularly, the

expression of differentiation markers on Ti-ASD treated ti-

tanium shows that genes related to the proliferation phase

(Coll I, Cbfa-1) were early expressed. Whereas genes

related to the mineralization phase (ALP, OPN, and BSP)

increased in a time-related way. Although SaOS-2 cells lost

the stringent control between proliferation and differentia-

Figure 8. Mineralization pattern, obtained with Alizarin Red S stain, of SaOS-2 cells grown on Ti,

Ti-ETC, and Ti-ASD (a) and the negative control samples of these surfaces without cells (b). [Color

figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]



tion22 when grown on Ti-ASD titanium, they showed a

progressive expression of genes associated with bone cell

differentiation similarly to the development sequence of the

normal osteoblasts.

The final differentiation parameter for osteoblast-like

cells is the formation of a calcified extracellular matrix;

related to biomaterials, the amount of mineralized matrix

formed on a material is the most reliable way to compare

the osteoinductive capacity of materials.27 Mineralization

occurred on all analyzed surfaces, but on Ti-ASD the num-

ber of bone-like nodules and the amount of mineralized

area was higher. This is in accordance with the results of

other authors11 who observed that Ti-ASD has an intrinsic

osteogenic activity because this surface may be recognized

by the cell as an environment being similar to that of a

bony tissue in its phase of repair for the high Ca/P ratio,

typical of newly deposited mineral phase.

In conclusion, Ti-ASD resulted to be a good surface for

osteoblast attachment and proliferation, also promoting the

maintenance of cell differentiation and matrix mineraliza-

tion, a fundamental requirement to sustain osseointegration

and the clinical success of dental implants.

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Attachment, proliferation and osteogenic response of osteoblast-like cells cultured on titanium...

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