Human gingival fibroblast functions are stimulatedby oxidized nano-structured titanium surfaces

Luigi Guida a, Adriana Oliva b, Maria Assunta Basile b, Michele Giordano c,Livia Nastri a, Marco Annunziata a,*aMultidisciplinary Department of Medical–Surgical and Dental Specialties, Second University of Naples, Via L. De

Crecchio, 6, 80138 Naples, ItalybDepartment of Biochemistry and Biophysics ‘‘F. Cedrangolo’’, Second University of Naples, Via L. De Crecchio, 7,

80138 Naples, Italyc Institute for Composite and Biomedical Materials, National Research Council (IMCB-CNR), Piazzale Enrico Fermi 1,

80055 Portici, Italy

j o u r n a l o f d e n t i s t r y 4 1 ( 2 0 1 3 ) 9 0 0 – 9 0 7

a r t i c l e i n f o

Article history:

Received 27 March 2013

Received in revised form

12 July 2013

Accepted 18 July 2013

Keywords:

Dental implant

Human gingival fibroblasts

Surface topography

Cell adhesion

Cell proliferation

Collagen synthesis

a b s t r a c t

Objectives: The aim of this study was to analyze the features of an oxidized titanium implant

surface and to evaluate its effects on the response of human gingival fibroblasts.

Methods: 10 mm � 10 mm � 1 mm turned (control) and oxidized (test) titanium samples

(P.H.I. s.r.l., Italy) were examined by scanning electron microscopy and atomic force

microscopy and characterized by height, spatial and hybrid roughness parameters. Primary

cultures of human gingival fibroblasts were seeded on titanium samples, and cell morphol-

ogy, adhesion, proliferation and extracellular matrix deposition, in terms of type I collagen

synthesis, were evaluated.

Results: Control and test surfaces appeared considerably different at the microscopic

analyses: turned samples were grooved, whereas oxidized surfaces showed a more complex

micro- and nano-scaled texture, as evidenced by roughness parameters. Cell adhesion and

proliferation rate, as well as collagen synthesis, were greater on oxidized vs turned surfaces.

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 and, mostly, hybrid parameters. Furthermore, oxidized surfaces enhanced

human gingival fibroblast adhesion, proliferation and extracellular matrix deposition, and

this could be due to the different structure at micro- and nano-scale levels.

Clinical significance: Oxidized nanostructured titanium surfaces could have a significant

clinical utilization in virtue of their affinity for soft tissue attachment at the implant neck

and/or at the transmucosal portion of the prosthetic abutment.

# 2013 Elsevier Ltd. All rights reserved.

Available online at www.sciencedirect.com

journal homepage: www.intl.elsevierhealth.com/journals/jden

1. Introduction

The use of implant-supported dental prostheses represents

today one of the most proposed rehabilitative option for the

treatment of edentulism worldwide, thanks to the positive

* Corresponding author. Tel.: +39 0815665515; fax: +39 0815665515.E-mail address: marco.annunziata@unina2.it (M. Annunziata).

0300-5712/$ – see front matter # 2013 Elsevier Ltd. All rights reservehttp://dx.doi.org/10.1016/j.jdent.2013.07.009

impact of such rehabilitations on oral function, and conse-

quently on systemic health, and to their high survival and

success rates over extended follow-up periods.1–6

The main cause of implant failure is represented by the

perimplantitis infection, as a consequence of the penetration

d.

j o u r n a l o f d e n t i s t r y 4 1 ( 2 0 1 3 ) 9 0 0 – 9 0 7 901

of bacterial plaque into the peri-implant sulcus,7,8 so that the

game of implant success is mainly played at level of the peri-

implant soft-tissue attachment. The interface between the

transmucosal portion of implant surface and the surrounding

soft tissue is characterized by a fibroblast-rich barrier tissue,

which represents the main obstacle to bacterial invasion of

deep peri-implant tissues, being of pivotal importance for the

long-term stability and maintenance of the implant itself.9–12

Topographic and physicochemical properties of implant

surfaces have been demonstrated to affect both hard and soft

peri-implant tissue responses. For instance, different surface

treatments are routinely used to increase implant roughness

and promote osseointegration.13 Conversely, the transmuco-

sal portion of implant rehabilitations is conventionally made

of a smooth turned titanium surface in order to minimize

bacterial colonization. However, there is a growing evidence

that implant surface characteristics may play a role in the

attachment of peri-implant soft tissues.12,14–16

One of the most recent frontiers in dental implant research

includes the modification of the surface topography at nano-

scale level. Nano-structured surfaces have been speculated to

influence cell behaviour in a different way compared to

conventionally sized surfaces. Nanoscaled features may

create biomimetic relationship between alloplastic surfaces

and bone tissue through the recapitulation of natural cellular

environments at the nanoscale level13,17,18 whereas little is

known about their effect on soft peri-implant tissues.

Anodic oxidation is one of the most diffused technology

able to create structures of nano-scale dimension on the

implant surface. Recently we have demonstrated that nano-

structured surfaces obtained by anodic oxidation can affect

bone cell behaviour.19 The aim of the present study was to

characterize the micro- and nano-texture of an oxidized

titanium implant surface in comparison with a conventional

turned one, and to evaluate the ability of such surface to affect

the response of human gingival fibroblasts (HGF) in terms of

adhesion, proliferation and collagenic matrix synthesis.

2. Materials and methods

2.1. Products and reagents

All cell culture biologicals 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.

2.2. Specimen preparation

Two different titanium implant surfaces were analyzed:

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 mm � 10 mm � 1 mm samples of commercially pure tita-

nium. Test samples were produced by a process of anodic

oxidation carried out for 24 h in an aqueous solution of 1 M

sulphuric acid and 0.15% hydrofluoric acid at a cell voltage of

20 V at room temperature. After anodization, the samples

were thoroughly rinsed in distilled water and subsequently in

acetone, and finally dried with nitrogen stream.

For cell culture assays the samples were sterilized by

autoclaving and put on the bottom of 24-well plates.

2.3. 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 microscopy (SEM) to visualize their topographic

features on a larger spatial range.

AFM technique is based on a tip of atomic level, which is

brought close to the sample. The interaction 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-dimensional map of outer

surface.

The images were obtained with an AFM-SNOM system: the

Multiview 1000 (by Nanonics Imaging Ltd), scanning probe

microscope operating in AFM tapping mode. The measuring

range available with this system was 75 mm 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 at a 50 mm � 50 mm dimensional

range. Six images were collected on different points, randomly

distributed upon the surface, belonging both to the centre, and

to the edge of the samples.

A 25 mm � 25 mm Gaussian filter was applied to separate

roughness from errors of form and waviness, as recom-

mended by Wennerberg and Albrektsson.20 The evaluation

and the images were obtained using SPIPTM (Scanning Probe

Image Processor, Image Metrology, Denmark) software. The

following surface parameters were considered:

Sa (mm) = average roughness; average height deviation

from a mean plane within the measuring area, Sds

(mm�2) = summit density; the number of summits per unit

area, Sdr (%) = developed interfacial area ratio; additional

surface area contributed by the roughness compared to a

totally flat plane.

2.4. Preparation of human gingival fibroblasts

Samples of gingival tissue were harvested from healthy

donors undergone periodontal surgery.

Informed consent and research protocol were institution-

ally approved, according to the Declaration of Helsinki.

Collected tissues were washed two times with phosphate

buffered saline (PBS; 150 mM NaCl, 20 mM sodium phosphate

pH 7.2) supplemented with antibiotics (100 U/ml penicillin,

100 mg/ml streptomycin) and cut into small pieces with a

sterile surgical blade. Tissue fragments were digested in 1 ml

of Dulbecco’s modified Eagle’s medium-F12 (DMEM-F12)

containing antibiotics and 1 mg/ml type IV collagenase

(Worthington Biochemical, Freehold, NJ, USA) at 37 8C for

3 h. Released cells were harvested, plated in complete DMEM-

F12 containing antibiotics and 10% foetal bovine serum, and

incubated at 37 8C in a 5% CO2 humidified atmosphere. First

cell islets were visible after 3–4 days and confluence was

Fig. 1 – 50 mm T 50 mm atomic force microscopy (AFM) 3D

images of oxidized and turned implant surfaces.

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reached in about 2–3 weeks. During this period, fresh medium

was added two times per week, never removing the entire

conditioned medium. After the confluence was reached, cells

were trypsinized and cultures expanded. For this study, HGF

obtained from two volunteers, one woman and one man, aged

35 and 56 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.

2.5. Cell adhesion and proliferation evaluation by MTT

Control and test samples were put on the bottom of 24-well

plates. HGF were seeded on implant surfaces at a density of

30,000 cells/cm2 in complete culture medium. Cell adhesion to

implant surfaces at 6 h and cell proliferation at 48 h and 7 days

from plating were assessed by MTT vitality assay.21 The key

component of this assay is 3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide (MTT). Mitochondrial dehydro-

genases of living cells reduce the tetrazolium ring, yielding a

blue formazan product that can be measured spectrophoto-

metrically. Cells were washed with PBS and incubated with

0.5 mg/ml MTT solution for 4 h at 37 8C. At the end of this time,

the liquid was aspirated and the insoluble formazan produced

was dissolved in isopropanol–HCl 0.1 M. The optical density

was measured at 570 nm, subtracting background absorbance

determined at 690 nm.

2.6. Cell adhesion and morphology evaluation by SEM andCLSM

Cell adhesion and morphology were also evaluated by

scanning electron microscopy (SEM) and confocal laser

scanning microscopy (CLSM). For SEM analysis cells were

plated on titanium surfaces as above mentioned. After 6 h the

cells were rinsed three times with PBS and fixed for 30 min

with 2.5% glutaraldehyde. The fixed cell layers were washed in

PBS, dehydrated by graded ethanol solutions (from 60% to

100%) and subjected to critical point drying. Samples were

mounted on stubs, coated with Au/Pd alloy and examined by

SEM (Philips SEM XL20).

Actin-based cytoskeleton was evidenced by immunofluo-

rescent staining using CLSM (C1-si, Nikon, Tokyo, Japan) after

24 h from cell seeding. The samples were prepared as follows:

titanium disks with layered cells were washed twice in PBS to

remove non attached cells. After rinsing, the cells were fixed in

4% paraformaldehyde in PBS for 10 min at 37 8C and

permeabilized with 0.2% Triton X-100 in PBS for 5 min at

37 8C. Permeabilized cells were treated with 0.5 ml/ml rhoda-

mine-phalloidin (Invitrogen, Paisley, UK) for 30 min and rinsed

in PBS.

2.7. Type I collagen synthesis

Conditioned medium of HGF cultured for 6 h, 48 h and 7 days

was collected and the amount of type I collagen released was

detected and quantified using a commercially available

enzyme-linked immunosorbent assay (ELISA) kit (Cosmo Bio

Co. Ltd, Tokyo, Japan). Assay buffer, sample and the

biotinylated anti-collagen antibody were added simultaneously

to the wells of microtiter-plate on which purified collagen was

immobilized. After washing, peroxidase labelled avidin, that

reacted with the biotinylated antibody on the microtiter-plate,

was added. Following further washing steps, tetramethylben-

zidine, a peroxidase substrate, was added. The level of type I

collagen in the sample was calculated on the basis of the optical

density determined at 450 nm and normalized to cell number.

In proportion to increase the collagen concentration of samples,

the colour density decreased since the amount of biotinylated

antibody and peroxidase labelled avidin decreased.

2.8. Statistical analysis

All the experiments were performed two times in quadrupli-

cate on two different cell preparations. No intra-group

difference was detected in any of the investigated parameters.

Differences between the experimental groups were analyzed

by non-parametric statistics (Wilcoxon Rank-Sum Test), with

the value of significance set at p < 0.05.22 Statistical analysis

was carried out using the NCSS software (NCSS for Windows,

Kaysville, Utah, USA).

3. Results

Three-dimensional AFM images of oxidized and turned

titanium samples are shown in Fig. 1. Fig. 2 shows SEM

images of the two surfaces at low and high magnification. It

is clearly observable in both SEM and AFM images that

grooves appeared on the turned samples, whereas the

oxidized ones showed a more complex topography. The

grooves present on the turned samples exhibited a prefer-

ential direction, whereas in the oxidized samples a uni-

formly rough surface, with grain-like particles of mean

diameter of 1.89 � 0.47 mm, seemed to be superimposed on a

previously grooved surface. A higher magnification SEM

image revealed the effective nanostructure of the oxidized

surface which was covered by tightly packed nano-tubules

with an external and internal mean diameter of 119 � 22 nm

and 50 � 11 nm, respectively.

Topographical parameters for both surfaces estimated by

AFM are reported in Table 1. When no filtering procedure was

applied, oxidized samples showed Sa values comparable to

turned ones, whereas using a 25 mm � 25 mm Gaussian filter

the situation reversed, with a significantly higher Sa mean

value for the oxidized surface. Such filter-dependence of the

Fig. 2 – Low and high magnification scanning electron microscopy (SEM) images of oxidized and turned titanium surfaces.

j o u r n a l o f d e n t i s t r y 4 1 ( 2 0 1 3 ) 9 0 0 – 9 0 7 903

roughness values was not evidenced for the other parameters

measured. The developed surface area of oxidized surface

showed the most evident difference between samples, being

the Sdr of the oxidized surface about one order of magnitude

higher than that of the turned one. On the contrary, no

significant difference of Sds between the two surfaces could be

assessed.

The following step in the characterization of titanium

surfaces was the biological evaluation of their interaction with

HGF in terms of cell adhesion, proliferation and extracellular

matrix deposition. At the SEM analysis, cells appeared

intimately spread on implant surfaces (Fig. 3). In particular

many cellular processes, such as philopodia and lamellipodia,

were evident on the oxidized surface, with a veil-like

appearance, in close contact with the underlying titanium

surface. Furthermore, the intimate interaction of cell process-

es with the nano-tubular structures became evident at higher

magnification.

Table 1 – Surface parameters of oxidized and turned titanium sGaussian filtering. Data are expressed as medians with minim

Parameter Sa (mm) Sd

Filter size None 25 mm � 25 mm None

Oxidized 0.227 (0.130–0.276) 0.076 (0.061–0.097) 1.105 (0.968–1.60

Turned 0.297 (0.193–0.343) 0.036 (0.020–0.69) 0.582 (0.405–1.70

Signif. p < 0.01

N = 6/group; signif. = statistically significant difference between oxidized

Immunofluorescence images (Fig. 4) showed a different

behaviour of the actin cytoskeleton of HGF grown for 6 h on

turned vs oxidized samples. In particular, cells appeared

fusiform and stretched out along the parallel grooves of the

turned surface, whereas a more evident spreading, without a

preferential direction, could be observed on the oxidized

sample.

When at 6-h MTT viability test was performed, markedly

greater values, as expression of higher numbers of adherent

cells, were evidenced on oxidized surfaces with respect to

turned ones (Fig. 5). Furthermore, at longer times, the

fibroblasts plated on the oxidized sample proliferated at a

substantially higher rate than cells attached on the turned

surface, reaching the maximum difference at 7 days.

In a parallel manner, when type I collagen production was

evaluated over time, HGF plated on the oxidized titanium

showed to synthesize a significantly higher protein amount,

normalized to cell number, compared with cells grown on the

urfaces (50 mm T 50 mm analysis range), with and withoutum and maximum values reported in parentheses.

s (mm�2) Sdr (%)

25 mm � 25 mm None 25 mm � 25 mm

0) 1.225 (1.090–1.700) 11.150 (7.860–24800) 10.500 (7.390–24.200)

0) 0.982 (0.769–1.910) 1.175 (0.552–3.050) 0.799 (0.212–2.480)

p < 0.01 p < 0.01

and turned.

Fig. 3 – Low and high magnification scanning electron microscopy (SEM) images of human gingival fibroblasts (HGF) grown

for 6 h on oxidized and turned titanium surfaces.

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turned surface, reaching a significant difference at 7 days

(Fig. 6).

4. Discussion

In this study, we tested in vitro if an oxidative titanium surface

treatment might positively affect the interaction between the

dental implant and the surrounding soft tissue, by evaluating

the adhesion and the vitality of human gingival fibroblasts

cultured on an oxidized implant in comparison with the

classical turned surfaces. Turned surfaces were chosen as

control because they represent the first and more extensively

implant surfaces tested, and the long-term success rate of

Fig. 4 – Confocal laser scanning microscopy (CLSM) immunofluo

gingival fibroblasts (HGF) grown for 24 h on oxidized and turne

rehabilitations realized with them continues to be the

reference point for all implant treatments.

A number of surface treatments of titanium samples for

dental implant applications have been successfully shown to

increase surface roughness in micro/nanometer range and to

enhance surface affinity for bone tissue, whereas only few

studies have investigated the possibility to ameliorate also the

affinity between implant surface and peri-implant soft tissue

by modulating the implant surface.

In particular, the enhancing of titanium surfaces’ bioactiv-

ity by nanostructuring technology represents nowadays a

growing aspect of the research in the dental implant field.

Nanostructured materials provide an increased surface area

and a better tissue-titanium contact rate.23 Furthermore, it has

rescence image of the actin-based cytoskeleton of human

d surfaces.

Fig. 5 – Human gingival fibroblast (HGF) adhesion and

proliferation on oxidized and turned titanium surfaces

assessed by MTT test. Data are expressed as medians with

lower/upper quartiles and minimum/maximum values.

N = 8/group; *p < 0.001.

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been speculated that they directly affect bone cell behaviour in

a different way from conventional rough surfaces, that is

creating a biomimetic relationship with host tissues through

the recapitulation of natural cellular environments at the

nanoscale level.13,17,24

When we talk of surface features in the nanometer scale,

we refer to topographical characteristics with dimensions less

than 100 mm. The oxidized surface we tested showed both a

micro- and a nanoscale topography. In particular, we

evidenced grain-like particles of microscale dimension super-

imposed on a previously grooved surface covered by tightly

packed tubular structures of nanoscale dimension.

Several approaches are currently available for manipulat-

ing experimental surfaces at the nanoscale level, including

self-assembly of molecules monolayers, physical approaches,

chemical approaches, nanoparticle deposition or optical

methods.13 All these technologies differ from one to another

for cost, time, reproducibility, complexity of the procedure and

for shape, size and chemistry of the nanostructures. The

anodic oxidation method is able to form nanostructured

features by creating a new oxide layer upon the surface of the

titanium implants used as an anode in a galvanic cell with a

suitable electrolyte.25,26 The characteristic of such oxide layer

can be modulated depending on the parameters used for

oxidation. The oxidized surfaces tested in our experiments

exhibited nanostructures of controlled and reproducible

dimension, with an extremely uniform and isotropic distribu-

tion over the surface.

Fig. 6 – Type I collagen synthesis by human gingival

fibroblasts (HGF) grown on turned and oxidized surfaces.

Data are expressed as medians with lower/upper quartiles

and minimum/maximum values. N = 8/group; *p < 0.001.

Although both tested oxidized and turned surfaces must be

included in the ‘‘smooth’’ category (following Albrektsson and

Wennerberg classification of 2004)27 basing on their Sa value at

a wide range, however they showed considerably different

characteristics at micro- and nanoscale level that could not be

detected by conventional roughness analysis. In fact, only

when a proper filter was applied to exclude errors of form and

waviness, and adjunctive three-dimensional surface param-

eters were considered, it was possible to find out the

significantly more complex texture of the oxidized surface

and its nanoscale features. This finding confirms what was

described by Wennerberg and Albrektsson20 that an exhaus-

tive characterization of a surface can be obtained only using

multiple roughness parameters and proper filtering proce-

dures. Each surface parameter, indeed, evaluates specific

topographical aspects and is expression of specific surface

features. For this reason, two distinct surfaces may appear

comparable in terms of a given surface parameter, but

significantly different measuring other ones or applying

specific filtering procedures. Also in our case, the surface

analysis of turned and oxidized surfaces led to considerably

different results in terms of Sa, for instance, with vs without

Gaussian filtering, or in terms of Sds vs Sdr. In particular, the

significantly higher Sdr values measured for the oxidized

surface with respect to turned one indicates a higher

developed area and is expression of the complex texture,

especially at nanoscale level, of such surface. At the same time,

however, both surfaces resulted comparable in terms of Sds,

which measures the density of summits over the surface. Such

finding on one side is expression of a similar pattern of summits

distribution over both surfaces and, on the other side, is also

affected by the specific thresholds existing for summits to be

properly detected, so that the oxidized surface could be

underestimated in terms of Sds. In fact, to be detectable and

measured as Sds, summits are constrained to be separated by at

least 1% of the minimum ‘‘X’’ or ‘‘Y’’ dimension comprising the

three-dimensional measurement area. Additionally, summits

are only found above a threshold that is 5% of the ten-point

mean roughness (Sz) above the mean plane.

The higher percentage of additional area contributed by the

complex nano-roughened oxidized surface could have some

implications from a biological standpoint at either tissue level,

providing larger and more intimate interlocking between

implant surface and peri-implant soft tissue, or cellular level,

as it will be discussed in succession.

In order to evaluate the biological features of these

surfaces, we choose, as cellular model, primary cultures of

human gingival fibroblasts, which are the main resident cell

population inside the peri-implant connective attachment.

We found that fibroblasts very intimately adhered on the

oxidized surface, taking contact with the complex micro and

nano topography of such surface by numerous philopodia-

like cellular extensions. Differently, HGF exhibited a less

intimate spreading on smooth turned surfaces, as evidenced

by comparing the respective SEM and immunofluorescence

microscopy images. Both direct cell–surface interactions

and indirect protein–surface interactions may explain this

finding,28 which is of pivotal importance, representing the

first step of every following event at the soft tissue-implant

interface.

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We also found that the adhesion and the proliferation rate

of fibroblasts was significantly increased on the oxidized

nano-structured surface with respect to the turned one, in a

time dependent manner.

Interestingly, and in support of our data, a recent study

demonstrated that fibroblast functions were stimulated by

oxidized nanostructured titanium surfaces,29 although in that

case cells of animal origin (NIH/3T3 murine fibroblasts) were

used.

Another finding of this study was that HGF cultured on

oxidized surfaces showed higher levels of type I collagen

synthesis. Type I collagen is the major component of the

extracellular matrix of connective tissues and plays a central

role in the architecture of the peri-implant tissue which is

formed by both epithelial and connective structures. In

particular, the connective attachment underlying the junc-

tional epithelium is constituted by a scar-tissue-like tissue

with high density of high turn-over fibroblasts (about one third

in volume) and collagen fibres (about two third in volume)

disposed in close contact with the titanium surface of the

implant neck.10

The more complex surface topography of anodic oxidized

samples at both the micro and the nano-scale level can

explain, at least in part, the different cellular functions put in

evidence in our study.

Different studies suggested that surface properties (topog-

raphy, physics, chemistry) of the transmucosal portion of

dental implants can affect the architecture of the peri-implant

mucosal barrier.12,15,16,30 It has been suggested that a certain

surface roughness may be needed for optimal soft tissue

sealing, enhancing the interaction between the surface

texture and soft tissue cell attachment and proliferation.30

In particular, minor epithelial downgrowth and longer

connective tissue seal have been histologically demonstrated

in humans for rough titanium implants (oxidized and acid-

etched) compared to smooth ones (turned).15

Surface roughness and chemical composition of the

implant surface, however, have been suggested to have a

significant impact on bacterial adhesion and plaque forma-

tion.31–33 It has been suggested by in vitro observations that a

surface roughness value (Ra) of about 0.2 mm can be consid-

ered as a threshold value below which no further significant

changes in the total amount of adhering bacteria can be

observed.34 However, the clinical relevance of such limit is

questionable, and the few and heterogeneous studies avail-

able do not allow to find a strong correlation between surface

characteristics and the initiation of peri-implantitis.7,35 Both

oxidized and turned surfaces tested in this study, although

characterized by not-filtered Sa values slightly higher than

0.2 mm, remain largely below the upper limit of smooth

surfaces (i.e. 0.5 mm), which are currently applied at the

transmucosal portion of fixtures and abutments. In this sense,

it is particularly interesting the possibility that nano-struc-

tured surfaces, may on one side enhance the attaching and

sealing of peri-implant soft tissues, being, on the other side, as

low-attractive for oral bacteria as conventional turned

surfaces.

In conclusion, the present in vitro study demonstrated that

a nanostructured titanium implant surface obtained by anodic

oxidation clearly stimulated gingival fibroblast functions over

time, including adhesion, proliferation and extracellular

matrix synthesis, compared to traditional smooth turned

surfaces. Such biological effects can be related to the different

texture between the two titanium surfaces at the micro- and

mainly at the nano-scale level, which could be properly

characterized only using a multi-parameter analytical ap-

proach.

The possibility to affect cell behaviour and, by this, increase

the stability of the peri-implant mucosal barrier, simply acting

on surface modifications is particularly fascinating. Further

studies, however, are needed to investigate the in vivo and

clinical implications of such encouraging preliminary results.

Acknowledgement

The authors want to thank P.H.I. (San Vittore Olona, Milano,

Italy) for kindly providing the titanium samples used in the

present study.

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