Foundation for Oral Rehabilitation

Evaluation of anodized
surfaces designed for
improved soft tissue
integration
Michael Nosswitz1, Misha Teale1, Stephanie Mathes1,
Andrea Venturato2, and Angelines Gasser2
1 Institute of Chemistry and Biotechnology, Zürich University of Applied Sciences, Wädenswil,

Switzerland
2 Nobel Biocare Services AG, Kloten, Switzerland
Correspondence to: stephanie.mathes@outlook.com
Published 12 March 2019

Abstract
A rapid and stable integration of dental implant systems into the surrounding tissues is achieved by enabling
migration, adhesion, proliferation and differentiation of the cells surrounding the implant and abutment surfaces.
Thus, a validation of abutment surface design requires a careful evaluation of their ability to provide a strong
attachment for the connective tissue and the epithelium. To evaluate the effect of surface anodization on soft tissue
integration, anodized (Xeal™) and machined surface titanium discs were used as culture substrates for humangingival epithelial cells (HGEPp) and primary human gingival fibroblasts (HFIB-G). The cellular response was
evaluated by quantifying cell growth, viability, development of extracellular matrix (collagen-I protein levels) andby assessing cell morphology. HFIB-G cells did not show any significant preference between the two surfaces
whereas HGEPp cells showed significantly faster growth on the Xeal™ surface. These results suggest that anodized
surface has the potential to accelerate healing and promote soft tissue health by increasing peri implant healing
and mucosa height.
Keywords: soft tissue, Xeal™, anodized surface, cell culture, keratinocytes, titanium

integration of the implant.6 The past years witnessed aremarkable effort aimed at the development of new or
improved materials with enhanced properties and able
to stimulate and support a better soft tissue adhesion. 6-

1. Introduction
Cellular behavior is strongly influenced by a material’s
surface chemical and physical properties (i.e.
hydrophilicity, stiffness, porosity, roughness, topography).1-3 In implant dentistry, titanium and its alloys
are widely applied due to their ability to support andpromote tissue integration while at the same time being
biologically inert.4-6
The rapid formation of a soft tissue seal at theabutment level represents one of the key requirements
to promote wound healing and to allow for a healthy

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When an implant system is placed, its surface
interacts with three main tissue types:•
The epithelia, formed by keratinocytes, which
form a tight seal separating the body from the outside
world. The cells are interconnected through cell-celljunctions that act as anchor points for actin filaments toprovide support points for the cytoskeleton.10
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Foundation for Oral Rehabilitation•
The connective tissue, formed by fibroblasts,
which are responsible for the production of new matrix
and glycoproteins.11, 12 This matrix secreted by the fibroblasts contributes to tissue adhesion to the implantsurface and its integration by mediating the interactions
with structural proteins such as fibronectin, vitronectin
and laminin.1•
The bone tissue, formed by osteoblasts, which
are responsible for the osseointegration of the implantby the formation of new bone.13
Cell adhesion to a surface is partially achieved throughfocal adhesion points, which consist of a high density ofprotein aggregates involved in mediating cell-cell andcell-material adhesion, migration, mechano-transduction
and signaling.14, 15 An inappropriate interaction with thesubstrate could inhibit cell division and ultimately
activate a pathway cascade leading to apoptosis.16
Hence, focal adhesion points are key players in cellproliferation and affect cell behavior by triggering
biochemical and biomechanical pathways. 1, 16 In this invitro study, anodized and nanostructured titanium
surface17 (Xeal™; Nobel Biocare AB, Göteborg, Sweden)
was compared with a standard, machined titanium
surface to evaluate their effect on cell adhesion andproliferation. To resemble the in-vivo conditions as
closely as possible, primary human gingival epithelial
cells (HGEPp) and primary human gingival fibroblasts(HFIB-G) were selected as test cell lines.

seeding. Laminin (Sigma-Aldrich, cat num L4544-100UL)
coating was performed according to manufacturer’s
guidelines.

2.3 Cell seeding
Cells were seeded on 6mm Ø discs at a concentrationof 10’000 cells/cm2. Per disc, 40μL of cell suspension
was pipetted leading to a drop sticking onto the surface.
Cells were allowed to settle for 4 hours in an incubator(37°C / 5% CO2). On laminin coated discs, cells seeding
was performed dispending 30μL of cell suspension. Next,
the discs were flushed with cell culture medium(2mL/well) and incubated until analysis was performed(37°C / 5% CO2).

2.4 Proliferation assay
Cell proliferation was assessed after 1, 3 and 6 daysby PrestoBlue® (Invitrogen, cat num A13262) assay
according to the manufacturer’s protocol. Defined cellconcentrations (1500, 3000, 6000, 12000, 25000,
50000, 100000 cells) were seeded into wells to enable
correlation to cell number. Using the linear regression fitof the standard curve the cell numbers for each disc was
calculated (n = 4).

a2. Materials and Methods
Two surfaces, machined without surface treatment as
control (machined) and anodized (Xeal, Nobel Biocare
AB), were produced from Ti alloy (Titanium-6 Aluminum4 Vanadium ELI) discs (diameter: 6 mm). Anodized
surface had regularly distributed nanostructures and anoxide layer approximately 150 nm thick.

2.1 Disc sterilizationb

Prior to cell seeding the discs were washed in EtOH
75% for 10min and three times in sterile ddH2O.
Subsequently, they were air-dried, sterilized under UVlight for 30min and distributed in 24 low adhesives wellplates (Thermo Scientific, cat num 12567104).

2.2 Disc coating
The two surfaces were divided into two groups andcoated respectively with FBS and laminin. FBS coatedsurfaces were prepared by incubation in 20% FBS
overnight (37°C, 5% CO2). After incubation, the discs
were washed 1x with PBS and air-dried prior to cell

Fig 1 a) Representative image of machined (left) and Xeal
(right) titanium discs. Scale bar: 1 mm. b) SEM image of themachined (left) and Xeal (right) surface. Scale bars: 1 µm.

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2.9 Picture acquisition

25

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The discs were analyzed on an inverted Olympus IX81
microscope. CellSens Dimension software version 1.6
was used to acquire fluorescent pictures and to takeoverview images for DAPI cell count.

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4

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Fold increase# of cells (103)Nosswitz et al

2.10 Statistical analysis
Statistical analysis was carried out with SigmaStat 3.5
software. One Way ANOVA was used to test fordifferences between the surface types. Significant
results were further analyzed by Tukey’s post-hoc test inorder to compare each surface type against every other.

1

0

0
1

3

6

Time (days)
Machined
Machined fold increase

3. Results and Discussion

Xeal
Xeal fold increase

The anodization process of titanium and its alloys has
been reported to induce physical and chemical changes
on the surface of the material.18 Visual inspection of the
Xeal surface showed a change of the surface color fromsilver to yellow (Fig 1a). Nanostructures could be readily
observed on the Xeal disc surface, suggesting an
increase of the total surface area (Fig 1b and 1c).

Fig 2 Mean HFIB-G cell number at 1, 3 and 6 days on
machined and Xeal surfaces. Right axis, fold increase of HFIBG cells over time normalized to the cell number at 1 day. Error
bars represent standard deviation.

2.5 Fixation

3.1 Effect of anodization on fibroblast behavior

The samples were washed 1x with PBS and then cells
were fixed by incubation with a 10% neutral bufferedformalin solution for 20min at RT. After incubation,formalin excess was removed by washing 3x with PBS.
Samples were stored at 4°C until further use.

The ability of fibroblasts to adhere to and grow on thetwo surfaces was assessed at 1, 3, and 6 days of cell
culture. At all evaluated timepoints, the total cell number
and the growth rate of HFIB-G cells were comparable
between the two surfaces (Fig 2). Similarly, the protein
levels of collagen-I, an indicator of fibroblast activity andtissue regeneration,19-21 were similar at all three time -2.6 DAPI quantification

Collagen-I [ng/1000cells]

Quantification of cell nuclei was performed by DAPI
staining. DAPI signal was read at 358 nm using 40x
microscope magnification. Cell nuclei were counted
automatically with the “count and measure” tool in
CellSens dimension 1.6 software.

2.7 Collagen-I protein levels
The determination of collagen production was
performed with a MicroVue™ CICP ELISA kit (Quidel, cat
num 8003) according to the manufacturer’s protocol.
Collagen-I amount was calculated per 1000 cells tonormalize the value to the cell population (n = 8).

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10
8
6
4
2
0
1

2.8 Focal adhesion staining

3

6

Time (days)

Fixed cells were permeabilized with 0.1% Triton X100, blocked for 30min in 1% BSA in PBS, incubated withprimary mouse α vinculin antibody (1:250) for 1h,
incubated with secondary Alexa Fluor 488 α mouse
antibody (1:400) and TRITC-conjugated phalloidin for
1h and subsequently stained for DAPI (300ng/mL).

Machined

Xeal

Fig 3 Mean amount of collagen-I per 1000 HFIB-G cells at
1, 3 and 6 days on machined and Xeal surfaces. Error bars
rep-resent standard deviation.

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the epithelial cells number on the two surfaces was evenbigger, suggesting Xeal surface could stronger promote
keratinocyte proliferation. Calculation of the foldincrease confirmed the faster cell growth on Xeal
surface. The small difference in the cell number at day 1
was likely related to the cells adapting to the new surface
and therefore having a low proliferation rate.
In addition to the assessment of cell viability,
quantification of cell numbers was performed through
DAPI staining at 1, 3 and 6 days. The results confirmed
the previous finding, showing higher total cell numberson the Xeal surface (Fig 5b). To evaluate to which extent
the observed differences between Xeal and machined
surfaces were induced by the sole properties of the Xeal
surface, a coating of the discs with either laminin or FBS

6 days (40x)

a

b

c

d

# of cells (x103)a

Fig 4 Triple fluorescent straining of HFIB-G cells for actin(red), DAPI (blue) and vinculin (green). a) and b) HFIB-G cells
on machined surfaces after 1 day and 6 days, respectively.
After 1 day focal contacts can be observed as small green dots
outlining the cells. c) and d) HFIB-G cells on Xeal surfaces after
1 and 6 days, respectively. Scale bars: 20 µm for a) and c),
100 µm for b) and d).

**

100

12
10

80

8

60

*

6

40

4

20

Fold Increase

1 day (40x)

2

0

0
1

3

6

Time (days)

points (all p>0.05); Therefore, the collagen-I levels were
not affected by the anodization process.
Next, the fibroblast morphology was analyzed throughtriple fluorescent staining of actin filaments, ascomponent of the cytoskeleton, vinculin, as mediator of
focal adhesion, and DAPI nuclear staining. Focal
adhesion sites were visible as small green dots outlining
the cells on both machined and Xeal surfaces (Fig 4a andc). After 6 days of cell culture, fibroblasts reachedconfluency and aligned radially along the grooves
present on the surfaces (Fig 4b and d). No majordifferences in fibroblast morphology were observed onmachined versus Xeal surfaces.

Machined

Xeal

Machined fold increase

Xeal fold Increase

Fluorescent area [%]

b
***

40
35
30
25
20
15
10
5
0

***

1

3

6

Time (days)

3.2 Effect of anodization on keratinocyte behavior

Machined

Following the fibroblast evaluation, keratinocyte
growth was assessed in the same cell culture model.
After the first day, no significant differences could beobserved between machined and Xeal surfaces (Fig 5a).
After 3 days, the cell number on Xeal surface wassignificantly higher than the cell number on machined
surface (Fig 5a), and after 6 days the difference between

Xeal

Fig 5 a) Mean HGEPp cell number at 1, 3 and 6 days on
machined and Xeal surfaces. Right axis, fold increase of HGEPp
cells over time normalized to the cell number at 1 day. b)
Quantification of the total DAPI fluorescent area of cells on
machined and Xeal surfaces at 1, 3 and 6 days. Error bars
represent standard deviation. Significance levels: p < 0.05 (*),
p < 0.01 (**) and p < 0.001 (***).

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4. Conclusions# of cells (103)120

The quantification of cell viability and cell number
confirmed that the Xeal surface has the ability toenhance proliferation of HGEPp cells. These results
corroborate previously reported studies whereanodization was shown to improve the interaction withlaminin, activate integrins and subsequently focaladhesion kinase (FAK) in epithelial cells, thus inducing a
proliferative cellular response.23 As expected, whenmachined and Xeal surfaces were coated with laminin or
FBS to mask chemical and physical differences between
the two surfaces, no major differences in cellproliferation were detected. Interestingly, HFIB-G cellproliferation, collagen-I protein levels and cell morphology were comparable between the two surfaces,
confirming that fibroblasts and keratinocytes havedifferent responses to certain surfaces. These data
confirm that the chemical and physical changes inducedby the Xeal layer are the principal mediators of theobserved differences in cell growth. This finding is
supported by scientific literature demonstrating how
surface chemistry, and nano-topographies can strongly
influence cell behavior.18, 23, 24 In conclusion, this study

100
80
60
40
20
0
1

3

6

1

Laminin

3

6

FBS
Time (days)

Machined

Xeal

Fig 6 (Left) Mean HGEPp cell number at 1, 3 and 6 days on
machined and Xeal surfaces coated with laminin. (Right) Mean
HGEPp cell number at 1, 3 and 6 days on machined and Xeal
surfaces coated with FBS. Error bars represent standard
deviation.

was performed. Laminin is a key component of the basal
membrane and is well known to provide excellent
support,22 while FBS is a standard coating used tosupport keratinocyte proliferation. HGEPp cells adhered
to and spread on both machined and Xeal surfaces
coated with laminin or FBS; however, no significant
differences in the cell number were observed at any of
the three timepoints (Fig 6) confirming that thepreviously described differences were indeed due to thechanges in surface properties of the two kinds of thetitanium discs.
A qualitative evaluation of cell morphology on the
different surfaces was performed through triple
fluorescent staining and showed cell attachment andformation of focal adhesion points on all surface types(Xeal and machined surfaces, uncoated and coated withlaminin or FBS) (Fig 7). Cell morphology looked very
similar: in all cases actin filaments were observed tosupportcell-substrateandcell-celladhesion.
Interestingly, actin filaments were evenly distributed
throughout the cell body (Fig 7, yellow arrows),
indicating active rearrangement of the cytoskeleton
suggesting that the cells were proliferating and migrating on the substrate surface. Vinculin proteins weredetected in focal adhesion points in all cases, suggestingthat the Xeal surface is a substrate able to support focal
adhesion attachment similarly to the control surface.

Xeal surface

Machined surface

b

c

d

e

f

Uncoated

Laminin coating

FBS coatinga

Fig 7 Triple immune-fluorescent straining of HGEPp cells for
actin (red), DAPI (blue) and vinculin (green). a) Xeal surface
and b) machined surface coated with FBS. c) Xeal surface andd) machined surface coated with laminin. e) Xeal surface andf) machined surface not coated. Scale bars: 20 µm.

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17. Milleret, V. et al. Rational design and in vitro
characterization of novel dental implant and abutment
surfaces for balancing clinical and biological needs. ClinicalImplant Dentistry and Related Research, epub ahead ofprint (2019).
18. Kim, K., Lee, B.-A., Piao, X.-H., Chung, H.-J. & Kim, Y.-J.
Surface characteristics and bioactivity of an anodized
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Injury 42, 551-555 (2011).
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23. Takebe, J., Miyata, K., Miura, S. & Ito, S. Effects of thenanotopographic surface structure of commercially puretitanium following anodization–hydrothermal treatment on
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24. Huang, H.-H., Wu, C.-P., Sun, Y.-S., Yang, W.-E. & Lee, T.H. Surface nanotopography of an anodized Ti–6Al–7Nb
alloy enhances cell growth. Journal of Alloys andCompounds 615, S648-S654 (2014).

suggests that anodization of titanium alloys has thepotential to improve proliferation of HGEPp cells while
maintaining the chemical and physical properties tosupport fibroblast proliferation and collagen-I production. Further studies have been conducted to confirm
the performances of the newly developed surface and togather a better understanding of the contribution of
surface chemistry and topography on the improved
tissue health.25, 26 Moreover, a recently reported outcome of a randomized controlled study on Xeal abutments showed consistently healthier mucosa incomparison to machined abutments.27

Conflict of interest
The authors declare no conflict of interest related tothis study. Andrea Venturato and Angelines Gasser are
currently employees of Nobel Biocare Services AG andparticipated in the study as contributing scientists.

Funding
Testing materials and reagents for the study were
provided and funded by Nobel Biocare Services AG.

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25. Susin, C., Fiorini, T., Demoner Ramos, U. & Schüpbach, P.
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Copyright© 2019 The Authors. Published by FOR.

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How to cite this article: Michael Nosswitz, Misha Teale,
Stephanie Mathes, Andrea Venturato, and Angelines
Gasser. Evaluation of anodized surfaces designed for
improved soft tissue integration. Foundation for Oral
Rehabilitation (FOR) 2019, pp. 1-7. www.for.org/scientificarticle/001

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