Download - Bone marrow mesenchymal stem cell response to nano-structured oxidized and turned titanium surfaces

Bone marrow mesenchymal stem cellresponse to nano-structured oxidized andturned titanium surfaces

Marco AnnunziataAdriana OlivaAntonietta BuoscioloMichele GiordanoAgostino GuidaLuigi Guida

Authors’ affiliations:Marco Annunziata, Agostino Guida, Luigi Guida,Department of Odontostomatological, Orthodontic andSurgical Disciplines, Second University of Naples,Naples, ItalyAdriana Oliva, Department of Biochemistry andBiophysics ‘‘F. Cedrangolo’’, Second University ofNaples, Naples, ItalyAntonietta Buosciolo, Michele Giordano, Institute forComposite and Biomedical Materials, NationalResearch Council (IMCB-CNR), Portici, Italy.

Corresponding author:Prof. Luigi GuidaDepartment of OdontostomatologicalOrthodontic and Surgical DisciplinesSecond University of NaplesVia L. De Crecchio6 – 80138 – NaplesItalyTel/fax: þ 39 081 566 5524e-mail: [email protected]

Key words: dental implant, fractal analysis, mesenchymal stem cells, oxidized titanium surface,

surface topography

Abstract

Objectives: The aim of this study was to analyse the topographic features of a novel nano-structured

oxidized titanium implant surface and to evaluate its effect on the response of human bone marrow

mesenchymal stem cells (BM-MSC) compared with a traditional turned surface.

Methods: The 10 � 10 � 1 mm turned (control) and oxidized (test) titanium samples (P.H.I. s.r.l.) were

examined by scanning electron microscopy (SEM) and atomic force microscopy (AFM) and

characterized by height, spatial and hybrid roughness parameters at different dimensional ranges of

analysis. Primary cultures of BM-MSC were seeded on titanium samples and cell morphology, adhesion,

proliferation and osteogenic differentiation, in terms of alkaline phosphatase activity, osteocalcin

synthesis and extracellular matrix mineralization, were evaluated.

Results: At SEM and AFM analyses turned samples were grooved, whereas oxidized surfaces showed a

more complex micro- and nano-scaled texture, with higher values of roughness parameters. Cell

adhesion and osteogenic parameters were greater on oxidized (Po0.05 at least) vs. turned surfaces,

whereas the cell proliferation rate was similar on both samples.

Conclusions: Although both control and test samples were in the range of average roughness proper

of smooth surfaces, they exhibited significantly different topographic properties in terms of height,

spatial and, mostly, of hybrid parameters. This different micro- and nano-structure resulted in an

enhanced adhesion and differentiation of cells plated onto the oxidized surfaces.

The principle of promoting osseointegration by

modification of implant surface features still

represents one of the most prolific and active

fields of dental implant research. Modern micro-

rough titanium surfaces has been widely im-

proved to promote implant osseointegration,

with respect to traditional smooth turned sur-

faces, both in terms of bone-to-implant contact

rate and retention into the bone (Buser et al.

1991; Ericsson et al. 1994; Gotfredsen et al.

2000; Cho & Park 2003; Shalabi et al. 2006).

The advantages of rough surfaces have been

confirmed also in the clinical field by long-term

data, particularly in the case of compromised

bone sites rather than in ordinary cases (Lambert

et al. 2009; Balshe et al. 2009).

Nevertheless, the research continues world-

wide with the aim to further improve the perfor-

mance of dental implants, accelerating and

maintaining their integration into hard and soft

tissues and/or extending their indications. One

of the most recent frontier in dental implant

research includes the modification of the surface

topography at a nano-scale level (for review

see Mendonca et al. 2008). Surface nano-

textures, providing an increased surface area

and finer surface roughness, may yield better

tissue-titanium mechanical interlocking (Meir-

elles et al. 2008). Furthermore, such nano-scaled

features have been speculated to directly affect

bone cell behaviour different from conventional

sized surfaces (Kubo et al. 2009; Guida et al.

2010), creating a biomimetic relationship be-

tween alloplastic surfaces and host tissues

through the recapitulation of natural cellular

environments at the nano-scale level (Mendonca

et al. 2008).

So far, only few studies have investigated the

importance of nano-scaled structures on implant

osseointegration but they mainly agree that such

structures have an impact on the early bone

healing, although their optimal size and distribu-

tion upon the implant surfaces is still far to be

defined (Wennerberg & Albrektsson 2009).

Date:Accepted 1 March 2011

To cite this article:Annunziata M, Oliva A, Buosciolo A, Giordano M, GuidaA, Guida L. Bone marrow mesenchymal stem cell responseto nano-structured oxidized and turned titanium surfaces.Clin. Oral Impl. Res. xx, 2011; 000–000.doi: 10.1111/j.1600-0501.2011.02194.x

c� 2011 John Wiley & Sons A/S 1

mailto:[email protected]

Despite the extensive clinical use of roughed

implants, indeed, the identity of the fundamental

parameters of implant surface topography that are

responsible for improving the rate and extent of

new bone formation remains largely unknown.

This is also due to the lack of standardized methods

and parameters inside the existing literature that

does not allow an easy comparison of the obtained

results. In 2000, Wennerberg and Albrektsson,

after a 10-year study on implant surfaces, formu-

lated a set of guidelines for their topographic

analysis highlighting the importance of reporting

multiple parameters and multi-scale measure-

ments for a proper surface characterization.

An ideal tool to investigate the interaction

between the bone cells and the implant surface

is represented by the bone marrow mesenchymal

stem cells (BM-MSC). BM-MSC are multipotent

cells which are able to self-renew and to differ-

entiate into precursors of several tissues, includ-

ing osteoprogenitor cells (Krebsbach et al. 1999;

Davies et al. 2002). They are involved in the

normal remodelling and reparative mechanisms

of bone, and play a central role in the osseointe-

gration process.

The aim of the present study was to character-

ize the micro- and nano-texture of a novel oxi-

dized titanium implant surface with respect to a

conventional turned one, and to evaluate the

ability of such surfaces to affect the response of

human BM-MSC in terms of adhesion, prolifera-

tion and osteogenic differentiation.

Materials and methods

Products and reagents

All cell culture biologics were purchased from

Gibco BRL (Grand Island, NY, USA), and all

chemicals were from Sigma Chemical Co. (St.

Louis, MO, USA), when not otherwise specified.

Specimen preparation

Two different titanium implant surfaces were

analysed: turned titanium surfaces (control) and

oxidized titanium surfaces (test). All specimens

were provided by a commercial firm (P.H.I. s.r.l.,

San Vittore Olona, Milano, Italy) in form of

10 � 10 � 1 mm samples of commercially pure

titanium. Test samples were produced by a pro-

cess of anodic oxidation in an aqueous solution of

1 M sulphuric acid and 0.15% hydrofluoric acid

at a cell voltage of 20 V at ambient temperature.

For cell culture assays the samples were

sterilized by autoclaving and put at the bottom

of 24-well plates.

Surface topography characterization

Qualitative and quantitative measurements of

titanium surfaces were made by atomic force

microscopy (AFM). In parallel, implant samples

were also imaged by scanning electron micro-

scopy (SEM) to visualize their topographic fea-

tures on a larger spatial range.

AFM technique is based on a tip of atomic

level, which is brought closer to the sample. The

interaction of the forces between the tip and the

sample are recorded by the deflection of a laser

beam reflecting on the cantilever attached to the

tip, in order to produce an accurate three-dimen-

sional map of the outer surface.

The images were obtained with an AFM-

SNOM system: the Multiview 1000 (by Nano-

nics Imaging Ltd, Jerusalem, Israel), scanning probe

microscope operating in AFM tapping mode. The

measuring range available with this system was

75mm in x, y and z direction. Super-thin probes

(cantilevered optical fibre probes, nominal spring

constant � 5 N/m, resonance frequency in the

range 50–100 kHz, by Nanonics Imaging Ltd)

with a tip radius of curvature 5 nm were used in

order to minimize convolution effects.

Images were acquired in four different dimen-

sional ranges of decreasing dimension:

50 � 50mm2 (range I), 15 � 15mm (range II),

5 � 5mm (range III) and 1.5 � 1.5mm (range

IV). For every dimensional range, seven images

were collected on different points, randomly dis-

tributed upon the surface, belonging both to the

centre, and to the edge of the samples. In this

way it was possible to obtain a multi-scale

characterization of the surface topography, from

standard length reported in literature down to the

length scale at which single cell interacts with

the surface.

A Gaussian filter was applied on large area

images (50 � 50 mm) to separate roughness from

errors of form and waviness, as recommended by

Wennerberg & Albrektsson (2000). The evalua-

tion and the images were obtained using SPIPt

(Scanning Probe Image Processor, Image Metrol-

ogy, H�rsholm, Denmark) software. The follow-

ing surface parameters were considered:

Sa (mm)¼ average roughness; average height

deviation from a mean plane within the measur-

ing area.

Sds (mm�2)¼ summit density; the number of

summits per unit area.

Sdr (%)¼developed interfacial area ratio; addi-

tional surface area contributed by the roughness

compared to a totally flat plane.

Sfd¼surface fractal dimension; the degree of

complexity of surface texture.

Preparation of human bone marrow mesenchymalstem cells (BM-MSC)

Samples of human bone marrow were harvested

from healthy donors, after informed consent was

provided, according to the Declaration of Hel-

sinki. Informed consent and research protocol

were institutionally approved. BM-MSC cultures

were initiated as described previously (Oliva et al.

2005). Briefly, heparinized bone marrow sample

was diluted 1 : 5 with complete culture medium

consisting of Opti-MEM containing 10% foetal

calf serum (FCS), 100 units/ml penicillin, 100mg/

ml streptomycin and 50 mg/ml sodium ascorbate,

and incubated at 371C in a 5% CO2 humidified

atmosphere. Although present in the bone mar-

row in a percent extremely low with respect to

the total of mononuclear cells, BM-MSC can be

easily obtained on the basis of their ability to

adhere to polystyrene plates, while the cells of

the haemopoietic lineage remain in suspension

and can be removed. After 48 h, the medium

containing all non-adherent cellular elements

was centrifuged for 10 min at 800 � g in order

to remove the haematopoietic cells, and added

again to the dish. In 3–4 days, several foci of

adherent spindle-like cells appeared and reached

the sub-confluence in 1–2 weeks. The medium

was refreshed every 3 days, each time leaving one

half of the conditioned medium. For this study,

BM-MSC obtained from two volunteers, one

woman and one man, aged 25 and 42 years,

respectively, were used. The cells harvested

from each donor were kept separately and not

pooled. Cultures between the second and fourth

passage were used in the present experiments.

Cell adhesion and proliferation

Control and test samples were put at the bottom

of 24-well plates. BM-MSC were seeded on im-

plant surfaces at a density of 15,000 cells/cm2 in

complete culture medium. Cell adhesion to im-

plant surfaces at 6 h and cell proliferation at 7 days

from plating were assessed by MTT vitality assay.

The key component of this assay is 3-(4,5-di-

methylthiazol-2-yl)-2,5-diphenyltetrazolium bro-

mide (MTT). Mitochondrial dehydrogenases of

living cells reduce the tetrazolium ring, yielding

a blue formazan product, which can be measured

spectrophotometrically. Cells were washed with

phosphate-buffered saline (PBS) and incubated

with 0.5 mg/ml MTT solution for 4 h at 371C.

At the end of this time, the liquid was aspirated

and the insoluble formazan produced was dis-

solved in isopropanol-HCl 0.1 M. The optical

density was measured at 570 nm, subtracting the

background absorbance determined at 690 nm.

Cell adhesion and morphology were also eval-

uated by SEM analysis. Cells were plated on

titanium surfaces as mentioned above. After 6 h

cells were rinsed three times with PBS and fixed

for 30 min with 2.5% glutaraldehyde. The fixed

cell layers were washed in PBS and dehydrated by

graded ethanol solutions (from 60 to 100%) and

critical point drying. Samples were mounted on

stubs, coated with Au/Pd alloy and examined by

Annunziata et al �BM-MSC response to oxidized and turned implant surfaces

2 | Clin. Oral Impl. Res. 10.1111/j.1600-0501.2011.02194.x c� 2011 John Wiley & Sons A/S

SEM (Philips SEM XL20, Eindhoven, the

Netherlands).

Osteogenic markers

The effects on cell differentiation were evaluated

analysing the expression of specific markers of

the osteoblastic phenotype, namely alkaline

phosphatase activity, osteocalcin production and

the mineralization of the extracellular matrix.

Alkaline phosphatase (AP) activity

The AP specific activity of BM-MSC grown on

the titanium surfaces was evaluated after 7 and

14 days of culture. Once removed the medium,

the wells were rinsed with 20 mM Tris HCl-

0.15 M NaCl, pH 7.4 (TBS) and the cells lysed

with a specific buffer (20 mM Tris/HCl, pH 7.4,

0.5 mM NaCl, 0.25%Triton X-100, 0.5 mM

PMSF, 0.5 mM DTT). AP activity was deter-

mined by measuring the release of para-nitrophe-

nol (PNP) from disodium para-nitrophenyl

phosphate (PNPP). The reaction mixture con-

tained 10 mM PNPP, 0.5 mM MgCl2, diethano-

lamine phosphate buffer pH 10.5, and 10–30mg of

cell lysate in a final volume of 100ml. After

10 min at 371C, the reaction was stopped by

adding 100ml of 0.5 M NaOH. PNP levels were

measured spectrophotometrically at 405 nm. The

AP activity was normalized to the protein con-

tent and expressed as units/mg protein, where

1 U was defined as the amount of enzyme that

hydrolyses 1 nmol of PNPP/min under the spe-

cified conditions

Osteocalcin synthesis

To evaluate osteocalcin synthesis, confluent cul-

tures, grown on the different surfaces for 2 weeks,

were incubated in FCS-free Opti-MEM in pre-

sence of 0.1% bovine serum albumin and

100 nM 1,25-dhydroxycolecalciferol for 48 h.

The levels of polypeptide secreted in the medium

were measured by means of an ELISA kit (Bio-

source International, Camarillo, CA, USA) that

utilizes highly specific monoclonal antibodies

and a peroxidase as a conjugated enzyme. The

amount of osteocalcin was calculated in ng/ml on

the basis of the optical density assessed at 450

and normalized to protein content.

Extracellular matrix mineralization

The ability of titanium surfaces to promote the

extracellular matrix mineralization was tested by

alizarin red staining (ARS). BM-MSC confluent

cultures were incubated for 21 days with an

osteogenic medium composed of 100 nM dexa-

methasone and 10 mM b-glycerophosphate.

Briefly, cell layers were fixed in 10% formalde-

hyde for 15 min before adding 1 ml of 40 mM

ARS (pH 4.1) per well. After a 20-min incuba-

tion, the layers were lysed with 10% acetic acid.

The lysates were then centrifuged at 20,000 � g

for 15 min and the supernatants neutralized with

10% ammonium hydroxide. Finally the samples

were read at 405 nm.

Statistical analysis

All the experiments were performed in quadru-

plicate on two different cell preparations. No

intra-group difference was detected in any of the

investigated parameters. Differences between the

experimental groups were analysed by non-para-

metric statistics (Wilcoxon Rank-Sum Test),

with the value of significance set at Po0.05.

Statistical analysis was performed using the

NCSS software (NCSS for Windows, Kaysville,

UT, USA).

Results

SEM images of the two different titanium sam-

ples at 1000 and 10,0000 magnifications are

shown in Fig. 1a and b, respectively. Figure 2

reports representative AFM height images of

control and test sample at different ranges. It is

clearly observable in both SEM and AFM images

that turned sample appear grooved, whereas the

oxidized one showed a more complex micro- and

nano-scaled topography. The grooves upon

turned sample exhibited a preferential direction

with their width and depth decreasing while

reducing the dimensional range: from 4.8 �0.9 mm large and 1.3 � 0.5 mm high in the range

I, to 468 � 43 nm large and 87 � 27 nm high in

the range IV. On the contrary, oxidized sample

shows a uniformly rough surface characterized

by micro-pits with a mean width value of

6.7 � 4.15mm, distributed over the whole

surface, superimposed on a previously grooved

surface.

High magnification SEM images (Fig. 1, panel

b) reveal the effective nano-structure of both

turned and oxidized surfaces. Turned surface at

nano-scale level was characterized by a uniform

granular aspect with nano-particles of mean dia-

meter of 39 � 0.9 nm. On the contrary, the

oxidized surface presents a complex structure

also at nano-scale level: it was covered by tightly

packed grain-like particles (mean diameter

89 � 16 nm) and nano-pores with an internal

mean diameter of 47 � 11 nm arranged in a

honeycomb pattern.

Topographical parameters estimated by AFM

images for such implant surfaces are reported in

Tables 1 and 2 and are discussed in detail below.

For the larger scale images (50 � 50mm) when no

filtering was applied, oxidized samples showed

significantly lower Sa values compared with

turned ones (Table 1). However, when a Gaus-

sian filter was applied, the situation reversed: Sa

was 91 nm for oxidized surface and 67 nm for

Fig. 1. Scanning electronic microscopy (SEM) images of turned and oxidized titanium surfaces at magnification x1000 (a) and

� 100000 (b).


c� 2011 John Wiley & Sons A/S 3 | Clin. Oral Impl. Res. 10.1111/j.1600-0501.2011.02194.x

turned one. Both Sa values belong to the nano-

scale level, but they are significantly different.

Such filter-dependence of roughness values was

not evidenced for the other parameters measured.

The developed surface area of oxidized surface is

about one order of magnitude higher than that of

turned surface. The fractal dimension (whose

theoretical value is between 2 and 3) of oxidized

surface was very high compared with turned one,

whereas no difference of Sds between the two

surfaces was verified at this length scale. Sa, Sdr

and Sfd values reflect the completely different

texture of the two surfaces as described at the

beginning of the paragraph on the basis of

the multi-scale AFM measurement reported in

Fig. 2.

Regarding the analysis performed at the other

three imaging scales, it is clearly observable from

Table 2 that while decreasing the dimensional

range, all surface parameters related to the oxi-

dized surfaces are always higher compared with

those of the turned ones, except for Sa values in

the range 15 � 15mm.

The following step in the characterization of

these surfaces has been the evaluation of the

effects of their interaction with the human BM-

MSC in terms of adhesion, proliferation and

osteogenic differentiation. The selection of these

cells is supported by their involvement into the

normal remodelling and reparative mechanisms

of bone, as well as in the osseointegration pro-

cess. BM-MSC were seeded at a density of

15,000 cells/cm2 and 6 h after plating cell adhe-

sion to implant surfaces was assessed by SEM

analysis and MTT viability test. Cells appeared

intimately spread on both turned and oxidized

surfaces: many intercellular interactions were

evident, in particular philopodia and lamellipo-

dia, with a veil-like appearance, were in close

contact with the underlying titanium surfaces,

the texture of which was clearly visible (Fig. 3,

panel a, white arrows). The intimate interaction

of cell processes with the surface texture at a

nano-scale level became evident at higher mag-

nification (Fig. 3, panel b).

When 6-h cell viability was measured, a

slightly higher number of cells (as expression of

MTT values) was evidenced on oxidized surfaces

(Po0.05) with respect to turned ones (Fig. 4). At

7 days, the MTT values became comparable

(P40.05) between test and control surfaces, in-

dicating that, after an early differential tendency

to adhere to the substrate, a comparable number

of cells attached successively on both surface

types reaching a similar proliferation rate.

Interestingly, both early and late osteogenic

markers were positively affected by test samples

compared with the control ones (Fig. 5). Alkaline

phosphatase specific activity is commonly con-

sidered an early marker of osteogenic differentia-

tion, being the AP, an enzyme implicated in the

formation of hydroxyapatite crystals during

extracellular matrix mineralization. Comparable

7-day values of AP specific activity between

cells grown onto test and control surfaces were

found, whereas, after 14 days, a significantly

(Po0.05) higher enzyme activity (about 40%)

for oxidized samples compared with turned ones

was evidenced.

This late trend for an enhanced osteogenic

differentiation of BM-MSC grown on oxidized

samples was confirmed by the expression of

Fig. 2. Atomic force microscopy (AFM) images of turned and oxidized samples at different analysis ranges.



greater levels (about 30%) of osteocalcin synth-

esis and a two-fold higher extracellular matrix

mineralization levels with respect to turned sur-

faces (Po0.05).

Discussion

The main finding of the present study has re-

garded the topographical modifications and the

biological performance induced by the oxidation

treatment on titanium implant surfaces. Indeed,

such treatment modified the topographical prop-

erties of titanium surfaces, at both micro- and

nano-scale level, and affected the biological re-

sponse of human BM-MSC to them compared

with control turned surfaces.

The oxidation treatment forms a thick oxide

layer on the titanium implants used as an anode

in a galvanic cell with a suitable electrolyte (Le

Guehennec et al. 2007; Wennerberg & Albrekts-

son 2009). From a micro-topographical point of

view, both oxidized and turned surfaces exhibited

Sa values o0.5mm. Following the classification

proposed by Albrektsson & Wennerberg (2004),

both of them must be considered in the same

category (i.e. smooth); however, their surface

texture appeared considerably different at either

micro or nano-scale level, as evidenced at both

microscopic (SEM) and roughness (AFM) ana-

lyses. In particular, the oxidized samples tested

in the present study were characterized by a more

complex surface texture, with micro-pits and

with nodules and pores of nano-scale dimension.

Such marked difference in terms of surface

texture between the control and test samples

was hardly detected by conventional roughness

analysis (i.e. Sa), which is solely descriptive of

height of surface irregularities, while it was

clearly evident taking into account spatial (i.e.

Sfd) and, mostly, hybrid (i.e. Sdr) roughness

parameters, which include also spatial informa-

tion. This finding highlights what was described

by Wennerberg & Albrektsson (2000) about the

obtaining of an exhaustive characterization of a

surface, that can be obtained only using multiple

roughness parameters and proper filtering proce-

dures. In this study, for instance, non-filtered Sa

values indicated the oxidized surface to be

smoother than the turned one. However, when

a proper filter was applied to exclude errors of

form and waviness, and also spatial and hybrid

parameters were considered, it was possible

to find out the significantly more complex tex-

ture of the oxidized surface and its nano-scale

roughness.

The higher percentage of additional area con-

tributed by the complex nano-roughened oxi-

dized surface could have some implications

from a biological standpoint at either tissue level,

providing larger and more intimate interlocking

between implant surface and bone, or cellular

level, as it will be discussed in succession.

In the present study, oxidized surfaces showed

to affect both cell interaction and behaviour

compared with turned samples. In particular,

BM-MSC adhesion was slightly but significantly

increased on oxidized surfaces. Both direct cell–

surface interactions and indirect protein-surface

interactions may explain this finding (Brunette

1988), which is of pivotal importance, represent-

ing the first step of every following event of the

osseointegration process at the bone–implant

interface.

Unlike cell adhesion, cell proliferation was not

conditioned by oxidized surfaces at 7 days of

culture. This finding indicates that, in an initial

phase after plating, cells adhere faster on the

oxidized rather than on the turned surfaces,

however, subsequently, a comparable number of

cells completes the adhesion process also on the

control sample and cells proliferate at a similar

rate on both surface types.

Furthermore, oxidized surfaces elicited the os-

teogenic potentiality of BM-MSC and induced

them to express at a higher level the parameters

typical of the osteoblastic phenotype. Indeed,

BM-MSC grown on test samples showed an

increase of alkaline phosphatase activity and

osteocalcin levels as well as of the mineralization

of the extracellular matrix, from 7 to 21 days

after plating.

Similar findings in terms of stimulation of cell

biological functions by nano-structured titanium

surfaces were reported also by other studies. Das

et al. (2009) showed enhanced adhesion, prolif-

Table 1. Surface parameters of turned and oxidized titanium surfaces (50 � 50 lm analysis range), with and without Gaussian filtering

Parameter Sa (mm) Sds (mm� 2) Sdr (%) Sfd

Filter size None 49 � 49 mm None 49 � 49mm None 49 � 49mm None 49 � 49mm

Oxidized 0.208 0.091 1.070 1.120 10.60 10.50 2.588 2.295(0.127–0.276) (0.075–0.113) (0.651–1.600) (0.692–1.260) (4.65–24.80) (4.48–24.70) (2.578–2.603) (2.260–2.361)

Turned 0.304 0.067 0.590 0.882 1.18 1.12 2.179 2.081(0.193–0.343) (0.046–0.093) (0.405–1.700) (0.634–1.810) (0.552–4.610) (0.287–4.010) (2.097–2.238) (2.013–2.109)

Signif. n n nn nn nn nn

N¼7/group.nPo0.05 andnnPo0.01 between oxidized and turned.

Data are expressed as medians with minimum and maximum values reported in parentheses.

Table 2. Surface parameters of turned and oxidized titanium surfaces at different analysis ranges

Parameter Range Sa (nm) Sds (mm� 2) Sdr (%) Sfd

Oxidized 15 � 15 mm 129.7 5.18 11.5 2.18(79.7–152.6) (4.31–5.95) (9.6–17.8) (2.16–2.23)

Turned 156 2.91 1.5 2.012(50.6–301.4) (2.58–2.99) (0.7–1.8) (2.007–2.023)

Significance nn nn n

Oxidized 5 � 5 mm 78.11 29.56 28.92 2.188(50.69–113.41) (28.23–33.41) (28.1–33.15) (2.115–2.269)

Turned 46.12 18.88 3.813 1.974(38.55–67.23) (14.29–19.97) (2.917–4.756) (1.910–2.007)

Significance nn nn nn n

Oxidized 1.5 � 1.5 mm 41.41 476 49 2.174(30.76–85.12) (378–567) (38–65) (2.091–2.263)

Turned 13.2 231 3.2 1.987(8.3–25.7) (168–261) (1.3–3.5) (1.929–2.076)

Significance nn nn nn n

N¼7/group.nPo0.05 andnnPo0.01 between oxidized and turned.

Data are expressed as medians with minimum and maximum values reported in parentheses.



eration, AP activity and mineralization by hu-

man osteoblasts cultured on oxidized implant

surfaces with nano-pores of 50–100 nm diameter.

Kubo et al. (2009) investigated implant surfaces

with a micropit-and-nano-nodule hybrid topogra-

phy reporting that the addition of nano-structures

to a micro-roughened implant surface was able to

stimulate the proliferation and the differentiation

of osteoblast cells.

In order to correctly interpret these findings,

different factors must be taken into account,

including the chemistry and the structural proper-

ties of the implant surfaces. Although both test

and control samples used in the present study

originated from the same type of commercially

pure titanium, it is noteworthy that every surface

treatment, such as anodization, affects both topo-

graphical and chemical properties of such materi-

als. In the opinion of the authors, the different

cellular response found between the two surfaces

may be mostly attributed to the complex surface

texture of the oxidized samples, with both a

micro- and a nano-scale feature, even if some

influence due to chemical surface alterations

cannot be excluded. Various reports support the

concept that nano- and micro-topography act

synergistically to promote implant osseointegra-

tion. In particular, it has been speculated that

nano-topography could play a role at cellular level

enhancing osteoblastic differentiation, without

directly affecting implant stability, which is pri-

marily conditioned by micro-scale topography

and implant macro-design (Mendonca et al.

2008). With respect to the old smooth surfaces,

the novel moderately rough implants are asso-

ciated with improved in vivo performances; many

of them show nano-scaled surface features (Meir-

elles et al. 2008; Guida et al. 2010), but whether

the nano-topography could have a role in such

improvement remains a matter of debate (Shalabi

et al. 2006; Wennerberg & Albrektsson 2009).

In conclusion, both oxidized and turned sur-

faces must be included in the ‘‘smooth’’ category

basing on their Sa value at a wide (50 � 50mm)

range, however their surface texture at micro and

nano-scale level was considerably different, and

could be properly characterized only using a

multi-range/multi-parameter approach and

proper filtering procedures. Oxidized surfaces

enhanced the adhesion of BM-MSC, as well as

their differentiation toward the osteoblastic phe-

notype, compared with the turned ones. Whether

these findings could be related to the topographi-

cal or to the physico-chemical changes induced

by the applied roughening technologies needs

supplementary analyses. Anyway, the possibility

to affect cell behaviour and, in turn, bone–im-

plant interaction and implant osseointegration,

acting on surface modifications, is particularly

fascinating.

Fig. 4. 6 hour-adhesion (a) and 7 day-proliferation (b) of BM-MSC cultured on turned and oxidized surfaces, as assessed by

MTT test. Data are expressed by box-plots indicating medians with lower/upper quartiles, and minimum/maximum values.

N=8/group; nnP o 0.01.

Fig. 3. SEM images of BM-MSC grown for 6 h on turned and oxidized samples at magnification x2,500 (a) and x100,000 (b).



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