Bone marrow mesenchymal stem cell response to nano-structured oxidized and turned titanium surfaces
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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: 39 081 566 5524e-mail: firstname.lastname@example.org
Key words: dental implant, fractal analysis, mesenchymal stem cells, oxidized titanium surface,
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 1mm 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; 000000.doi: 10.1111/j.1600-0501.2011.02194.x
c 2011 John Wiley & Sons A/S 1
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-
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.
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 puretitanium. 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 therange 50100 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 (rangeIV). 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
A Gaussian filter was applied on large area
images (50 50 mm) to separate roughness fromerrors 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, Hrsholm, Denmark) software. The follow-
ing surface parameters were considered:
Sa (mm) average roughness; average heightdeviation from a mean plane within the measur-
Sds (mm2) summit density; the number of
summits per unit area.
Sdr (%)developed interfacial area ratio; addi-tional surface area contributed by the roughness
compared to a totally flat plane.
Sfdsurface fractal dimension; the degree ofcomplexity 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 orderto remove the haematopoietic cells, and added
again to the dish. In 34 days, several foci of
adherent spindle-like cells appeared and reached
the sub-confluence in 12 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-
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
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 1030mg 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-
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-