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

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

Post on 21-Jul-2016




2 download

Embed Size (px)


  • 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:

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

    surface topography


    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-

    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 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

    the surface.

    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-

    ing area.

    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-

    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


    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 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-

    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-