Int. J. Oral Maxillofac. Surg.

2009; 38: 274278

doi:10.1016/j.ijom.2008.12.016, available online at http://www.sciencedirect.com

Research Paper
Dental Implants

In vivo low-density bone I. Braceras1,4, M. A. De Maeztu2,

J. I. Alava1, C. Gay-Escoda3
1
2
Inasmet-Tecnalia, San Sebastian, Spain;
Private Practice, Tolosa, Spain; 3University of
apposition on different implant Barcelona Dental School, Spain; 4Lifenova
Biomedical, Spain

surface materials
I. Braceras, M. A. De Maeztu, J. I. Alava, C. Gay-Escoda: In vivo low-density bone
apposition on different implant surface materials. Int. J. Oral Maxillofac. Surg. 2009;
38: 274278. # 2009 International Association of Oral and Maxillofacial Surgeons.
Published by Elsevier Ltd. All rights reserved.

Abstract. During osseointegration, new bone may be laid down on the implant surface
and/or on the old bone surface; the former is known as contact osteogenesis and the
latter as distance osteogenesis. Implant surface topography and material
composition affect this process. The present study evaluates Ca and P apposition
onto three different dental implant material surfaces (carbon monoxide (CO) ion
implantation on Ti6Al4V, sand blasting and acid etching on commercially pure
titanium and untreated Ti6Al4V) on the mandibles of beagles after healing periods
of 3 and 6 months. Energy dispersive spectroscopy is useful for identifying low-
density bone relative to surrounding mature bone, allowing for discrimination of the
Keywords: dental implant; ion implantation;
osteogenesis source. Low-density bone was only found at the apical end; there was
energy dispersive spectroscopy; calcium; os-
none on the surface of untreated implants. Low-density bone arising from mature teogenesis; XPS.
bone towards the implant at month 3 (i.e. distance osteogenesis) was only present on
the CO ion implanted samples, due to the modification of the surface nano- Accepted for publication 22 December 2008
topography and the chemistry and structure of the material. Available online 5 February 2009

Success in treating patients with dental
implants has lead to more than one million
implant placements per year worldwide.
The challenge of achieving fast and
dependable functionality of implant-sup-
ported teeth remains. Research is being
carried out to establish safe early loading
times for individual patients, and to
achieve faster intimate implant bone
bonding (osseointegration) so that masti-
cating loads can be applied without risk of
implant failure. The implant surface plays
a critical role in osseointegration.2 New
bone may be laid down on the implant
surface and/or on the old bone surface
around endosseous implants, the former
is known as contact osteogenesis and the
latter as distance osteogenesis.8,23
With distance osteogenesis, the old
bone may provide osteogenic cells that
lay down a new matrix, so that the implant
surface becomes surrounded by bone. Dis-
tance osteogenesis is typically expected in
cortical bone and explains the osseointe-
gration of machined-metal implants. In
contrast, with contact osteogenesis new
bone first forms on the implant surface.
The surface first has to be colonized by
bone cells, so that bone matrix synthesis
can begin. This results in de novo bone
apposition on the implant surface, while
bone remodelling occurs at discrete sites.8
Optimizing contact osteogenesis by
implant surface design to ensure early
stability is of great importance, especially
for III and IV type bone,21 which may not
offer sufficient cortex to provide stability.
In these cases, the recruitment of osteo-
genic cells to the implant surface and

0901-5027/030274 + 05 $30.00/0 # 2009 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

subsequent bone formation is the only way
in which implant stability can be
achieved.9 Newly formed peri-implant
bone has been observed on implants
inserted in poor bone sites in patients.19
Early biological fixation may allow the
time before loading the implant to be
reduced, favouring early or immediate
implant loading. This initial trabecular
bone is gradually replaced by the mature
lamellar bone that characterizes implant
osseointegration.16
The composition and roughness of the
implant surface affect the cellular
response to the implant material.20
Increased surface roughness of dental
implants has demonstrated greater bone
apposition. The effect of modifying sur-
face chemistry remains unknown.7
The present study will look for evidence
of contact osteogenesis occurring at three
different implant surfaces. It will try to
establish a relationship between osteogen-
esis and surface properties. In the follow-
ing samples, either the surface micro-
topography or the surface chemistry (plus
nanotopography) have been modified: an
untreated machine-turned implant, a sand-
blasted and acid-etched implant surface,
and a surface implanted with carbon mon-
oxide (CO) ion. A surface implanted with
CO ion induces a signalling pathway that
promotes the differentiation and apposi-
tion of osteoblasts5 that leads to more
complete osseointegration.4,10
This study aimed to identify low-den-
sity bone tissue relative to surrounding
mature bone, on and around sections of
dental implants tested in vivo in the jaw-
bones of beagles. This was achieved
through energy dispersive spectroscopy
(EDS), which has proved a useful techni-
que.
Materials & methods
Implant materials
Three different implant surface materials
were considered in this study: machined
Ti6Al4 V ELI (ASTM F136), in a Spli-
Table 1. Percentage of low bone density occurrence on ion-implanted (i.i.), sand-blasted and acid-etched (SLA1) and untreated (Ctrl) dental
implants.
Time Surface
from implant
3 months i.i. 20%
SLA 25%
Ctrl 0%
6 months i.i. 25%
SLA 0%
Ctrl 0%
In vivo low-density bone apposition on implant surface materials
neTwist1 dental implant (Zimmer Dental
Inc., Carlsbad, CA, USA), which was used
as a control sample; sand-blasted and acid-
etched (or SLA1) commercially pure
grade IV titanium (ASTM F67) (Institut
Straumann, Waldemburg, Switzerland);
CO ion implanted Ti6Al4 V ELI (ASTM
F136), in a SplineTwist1 dental implant
(Zimmer Dental Inc., Carlsbad, CA,
USA).
The first group of implants was used as a
control, and presented an average Ra of
0.4 mm. In the second group, the sand
blasting was performed with corundum
followed by acid etching with a mixture
of HCl/H2SO4, with a resulting chemical
composition of the surface of TiO2. The
topography consisted of craters, approxi-
mately 30100 mm in diameter, overlaid
with pits 13 mm in diameter, with an
overall Ra of 45 mm.3
In the latter case, the implants were
subjected to CO ion implantation surface
treatment using a Danfysik Model 1090
high-current implanter (Jyllinge, Den-
mark), at low temperature (<170 8C).
The ion implantation surface treatment
modified the outermost surface properties,
such as the nanotopography, chemistry
and surface energy. The surfaces pre-
sented an average nanotopography of 2
2.6 nm Ra6 on top of the microtopography
found in the machined samples used as
controls. The surface chemistry was ana-
lysed using X-ray photoelectron spectro-
scopy (XPS) in a VG spectrometer model
Microlab MKII, where C, Ti, Al and O
spectra were obtained at a pass energy of
50 eV and energy interval of 0.1 eV per
step.
Animals & surgical procedure
The design of the study was approved by
the Ethics Committee on Animal Research
at the University of Barcelona, Spain, and
was performed following ISO-10993-2
and ISO-10993-6 standards.
Six samples per surface material type
and healing period were obtained from a
previous in vivo test and used in this study.
Low density hard tissue
from bone to implant between bone and implant
40%
0%
0%
50%
33%
20%
275
That study had aimed at quantifying the
bone implant contact percentages of six
different implant surfaces/groups, which
stayed in the jawbones of adult beagles for
3 and 6 months. Results and the surgical
procedure are reported elsewhere11,12. In
the first step of surgery, three mandible
premolars were removed. Three months
later, tartar was removed from the remain-
ing teeth 2 days prior to intervention and
the implants were placed in the previously
edentulated zones, following approved
anaesthetic and analgesic protocols. In
the insertion surgery, six implants (one
from each study group) were implanted
submerged in each animal, with three on
each side, following a homogeneous
mesial /distal distribution of the different
groups.
The animals were sacrificed 3 and 6
months after placement of the implants
following sedation. Bone blocks contain-
ing the implants were extracted and
immersed in 4% formalin solution until
laboratory processing.
Histological preparation and EDS
Microscopic evaluation using environ-
mental scanning electron microscopy
(ESEM) and EDS was carried out directly
on blocks of bone tissue and implant
embedded in resin, preceded by sectioning
and polishing1, and deposition of a thin
layer of gold, using a JEOL JSM-
59110LV (Akishima City, Tokyo, Japan)
electron microscope and an OXFORD
INCA-300 (Witney, Oxon, UK) EDS.
Low calcium and phosphorus density
bone, relative to mature bone, was identi-
fied with EDS around the section peri-
meter of each of the implants. Whether
the low-density bone tissue was isolated or
connected to the surface of the implant or
mature bone was ascertained. The low-
density bone was then classified into four
different categories: low-density bone on
the implant surface; low-density bone on
mature bone; low-density bone between
mature bone and implant; and low-density
bone among mature bone.
among bone
40% 0%
75% 0%
0% 25%
75% 25%
100% 0%
40% 100%
276 Braceras et al.

Fig. 1. Ca, P, C and Ti line profiles by EDS at the surface of CO ion implanted dental implant surface at month 3.

Statistical analysis treatment far exceeded the naturally
2 occurring oxide layer. Different titanium
The x test was used for data analysis. A p-
oxides and carbides were present at the
value of less than 0.05 was considered
affected surface, but CC and CO bonds
significant.
were also detected together with C-rich
compounds.
Ca, P, Ti and C line profiles of the bone
Results
surrounding the implant at sites along the
The surface chemistry of the CO ion contact perimeter were then obtained by
implanted samples was studied by XPS, EDS. Results obtained for the samples,
which showed a profile of C and O present which stayed in the jawbones of the dogs
beyond 50 nm in depth. The surface layer for 3 and 6 months, are shown in Table 1
affected by the ion implantation surface and Figs. 1 and 2.
At month 3, the control group showed
no low-density bone in any of the sam-
ples at the implant surface arising from
mature bone, or between implant and
mature bone. The sand-blasted, acid-
etched and CO ion implanted groups
presented samples with low-density
bone at both the implant surface and
in the region between mature bone and
the implant. Low-density bone was
observed arising from mature bone
towards the implant in CO ion implanted
samples.

Fig. 2. Ca, P, C and Ti line profiles of low-density and mature bone by EDS on the surface of a CO ion implanted dental implant surface at month 6.

At month 6, no low-density bone was
observed at the surface of any of the
samples from the control untreated group,
the sand-blasted group or the acid-etched
group. Low-density bone arising from
mature bone was observed in the three
groups, but only in the CO ion implanted
group did some samples show low-density
bone at the implant surface.
Overall, low-density bone apposition at
the implant surface presented an average
thickness of around 40 microns at month 3
(Fig. 1) and 70 microns at month 6 (Fig. 2).
Discussion
Previous to the EDS analysis, all of the
samples containing a section of a dental
implant together with the surrounding
bone, had been analysed using ESEM,
resulting in a bone implant contact per-
centage (%BIC) of 61  8%, 33  20%
and 46  18% at month 3 and 62  2%,
49  9% and 52  8% at month 6, for the
CO ion implanted, untreated control and
SLA1 dental implants, respectively.11,12
Line profile analysis and elemental
mapping by EDS makes it possible to look
at site-to-site variations, and provides
information on the geographic distribution
of calcium to phosphorus ratios and on the
presence of trace elements in bone when
appropriate corrections and instrumental
calibrations are included.27
Spectroscopic mapping of calcium has
been used to study soft tissue, such as
nervemuscle18 and endoplasmic reticu-
lumGolgiexocytic membrane systems,24
as well as hard tissue. In the latter case,
several studies have looked at bone forma-
tion on biomaterials using ESEM and EDS.
It has been found that bone tissue does not
bond directly to CaSiO329 or to alkali mod-
ified implants28 but through a Ca/P layer.
The effect of sintered silicate substituted
hydroxyapatite on the remodelling pro-
cesses at the bone implant interface has
been analysed.25
Ca and P mapping of bone and bioactive
bone cement interfaces is useful for study-
ing the different response to cancellous
and cortical bone22 and in assessing the
chemical and biological integration of
mouldable bioactive ceramics.14 EDS
mapping images have been used to quan-
tify ingrown bone into a porous material
using digital image processing15. The
amount of Ca and P formation on micro-
phase compared with nanophase implan-
table metallic alloys cultured with and
without osteoblasts has also been studied,
demonstrating that the nanophase has an
influence, with increased Ca and P deposi-
tion in the case of nanophase Ti6Al4V26.
In vivo low-density bone apposition on implant surface materials
This result is in accordance with the idea
that local topography is an important fac-
tor in the location and timing of bone
formation in vitro and in vivo in normal
bone turnover, fracture repair and the
incorporation of bone grafts.17
Histomorphometric measurements
reveal that bone ingrowth reaches a pla-
teau after 4 weeks of initial healing on
porous surfaced titanium alloy dental
implants in dogs13. This has been con-
firmed by part of the present study
reported elsewhere11,12, where it was
observed that osseointegration reached a
plateau at month 3 for the CO ion
implanted and SLA1 surfaces, but not
before month 6 for the untreated
machine-turned implants.
In the EDS analysis, all of the low Ca
and P density tissue was found at the
apical lower end of the implants. This
confirms the idea that healing patterns in
cortical and trabecular bone are different;
the latter relying on contact osteogenesis
and therefore being better adapted to rapid
healing than cortical bone.9 No low-den-
sity Ca/P apposition was observed at the
surface of machine-turned implants
(Table 1), confirming that bone apposition
is favoured by surface modification. More
low-density bone was identified around
the machine-turned control implants at
month 6 than at month 3, indicating that
osseointegration was still underway.
As only one section of the implant bone
interface per implant was analysed by
EDS, it could not be clearly defined
whether the low-density bone identified
between bone and implant or among
mature bone was coming at from the
implant surface, mature bone or else-
where.
While low-density bone arising from
mature bone and oriented towards the
implant at month 6 was present in all three
surfaces, this was only true for CO ion
implantation at month 3, indicating that
modification of the titanium surface chem-
istry may promote early distant osteogen-
esis.
This surface modification may render
the surface of the implant active for a
longer period of time. Low-density bone
was observed at month 6 on the CO ion
implanted batch, unlike the SLA1 and
untreated surfaces. As the implants had
a randomised distribution, animals with
low-density bone detected at the SLA1
implant also presented low-density bone at
the CO ion implanted implant, suggesting
a patient-dependent pattern.
Low-density bone presence was more
frequent at the CO ion implanted and
SLA1 surfaces than on the control sam-
277
ples, with the difference being statistically
significant for low-density bone coming
from bone to implant (p = 0.001) for CO
ion implantation relative to control sam-
ples.
In conclusion, EDS is a useful technique
for identifying low-density bone relative
to surrounding mature bone, and thus dis-
criminating distance and contact osteo-
genesis. This technique provides
valuable information about the influence
of the implant surface topography and
surface chemistry on the osteogenic pro-
cess. This study supports the conclusion
that topography and chemical modifica-
tions may promote osteogenesis and allow
contact osteogenesis to occur.
In the case of CO ion implantation, this
may be due both to the nanotopography and
the surface chemistry, which presents a
variety of meta-stable C-rich compounds.
These readily react with the surrounding
biomolecules once implanted, eliciting a
response that promotes the differentiation
and apposition of osteoblasts that leads to
more complete osseointegration.
Acknowledgements. The authors would
like to express their gratitude for the
financial assistance provided by the Span-
ish Ministry of Science and Technology
CDTI- under Project n.20020327.
References
1. Alava JI, Marn F, Intxaurrandieta
A, Braceras I, De Maeztu MA. Simul-
taneous materialographic and histological
thin section preparation: Investigation of
bone integration on dental implants.
Structure Struers J Materialography
2005: 6: 16.
2. Albrektsson T, Branemark PI, Hans-
son HA, Lindstrom J. Osseointegrated
titanium implants. Acta Orthop Scand
1981: 52: 155170.
3. Boyan BD, Schwartz Z. Response of
musculoskeletal cells to biomaterials. J
Am Acad Orthop Surg 2006: 14: 157.
4. Braceras I, Alava JI, Onate I, Bri-
zuela M, Garca-Luis A, Garagorri
N, Viviente JL, De Maeztu MA.
Improved osseointegration in ion implan-
tation treated dental implants. Surf Coat
Tech 2002: 158-159: 2832.
5. Braceras I, Onate JI, Goikoetxea L,
Viviente JL, Alava JI, De Maeztu
MA. Bone cell adhesion on ion implanted
titanium alloys. Surf Coat Tech 2005:
196: 321326.
6. Braceras I, Alava JI, Goikoetxea L,
De Maeztu MA, Onate JI. Interaction
of engineered surfaces with the living
world: ion implantation vs. osseointegra-
tion. Surf Coat Tech 2007: 201: 8091
8098.
278 Braceras et al.
7. Buser D, Broggini N, Wieland M,
Schenk RK, Denzer AJ, Cochran
DL, Hoffman B, Lussi A, Steinemann
SG. Enhanced bone apposition to a che-
mically modified SLA titanium surface. J
Dent Res 2004: 83: 529533.
8. Davies JE. Mechanisms of endosseous
integration. Int J Prosthodont 1998: 11:
391401.
9. Davies JE. Understanding Peri-Implant
Endosseous Healing. J Dent Educ 2003:
67: 932949.
10. De Maeztu MA, Alava JI, Gay-
Escoda C. Ion Implantation: Surface
treatment for improving the bone integra-
tion of titanium and Ti6Al4V dental
implants. Clin Oral Impl Res 2003: 14:
5762.
11. De Maeztu MA, Braceras I, Alava JI,
Sanchez MA, Gay-Escoda C. Histo-
morphometric study of ion implantation
and Diamond Like Carbon (DLC) as
dental implant surface treatments in Bea-
gle dogs. Int J Oral Maxillofac Implants
2007: 22: 273279.
12. De Maeztu MA, Braceras I, Alava JI,
Gay-Escoda C. Improvement of osseoin-
tegration of titanium dental implant sur-
faces modified with CO ions: a
comparative histomorphometric study in
beagle dogs. Int J Oral Maxillofac Surg
2008: 37: 441447.
13. Deporter DA, Watson PA, Pilliar
RM, Melcher AH, Winslow J, How-
ley TP, Hansel P, Maniatopoulos C,
Rodriguez A, Abdulla D. A histologi-
cal assessment of the initial healing
response adjacent to porous-surfaced,
titanium alloy dental implants in dogs. J
Dental Res 1986: 65: 10641070.
14. Engqvist H, Schultz-Walz JE, Loof
J, Botton GA, Mayer D, Phaneuf
MW, Ahnfelt NO, Hermansson L.
Chemical and biological integration of
a mouldable bioactive ceramic material
capable of forming apatite in vivo teeth.
Biomaterials 2004: 25: 27812787.
15. Erli HJ, Ruger M, Ragob C, Jahnen-
Dechent W, Hollander DA, Paar O,
von Walter M. The effect of surface
modification of a porous TiO2/perlite com-
posite on the ingrowth of bone tissue in
vivo. Biomaterials 2006: 27: 12701276.
16. Franchi M, Fini M, Giavaresi G,
Ottani V. Peri-implant osteogenesis in
health and osteoporosis. Micron 2005: 36:
630644.
17. Gray C, Boyde A, Jones SJ. Topogra-
phically induced bone formation in vitro:
Implications for bone implants and bone
grafts. Bone 1996: 18: 115.
18. Grohovaz F, Bossi M, Pezzati R, Mel-
dolesi J, Tarelli FT. High resolution
ultrastructural mapping of total calcium:
Electron spectroscopic imaging/electron
spectroscopy analysis of a physically/
chemically processed nerve-muscle pre-
paration. Proc Natl Acad Sci U S A 1996:
93: 47994803.
19. Iezzi G, Degidi M, Scarano A, Perroti
V, Piattelli A. Bone response to sub-
merged, unloaded implants inserted in
poor bone sites: a histological and histo-
morphometrical study of 8 titanium
implants retrieved from man. J Oral
Implantol 2005: 31: 225233.
20. Kieswetter K, Schwartz Z, Dean DD,
Boyan BD. The role of implant surface
characteristics in the healing of bone. Crit
Rev Oral Biol M 1996: 7: 329345.
21. Lekholm U, Zarb GA. Tissue-integrated
prostheses: osseointegration in clinical
dentistry. Chicago: Quintessence 1985:
p. 199.
22. Ni GX, Coi YS, Lu WW, Ngan AHW,
Chiu KY, Li ZY, Tang B, Luk KDK.
Nano-mechanics of bone and bioactive
bone cement interfaces in a load-bearing
model. Biomaterials 2006: 27: 1963
1970.
23. Osborn JF, Newesely FH. Dynamic
aspects of the implant-bone interface.
Munchen Carl Hanser Verlag 1980:
111123.
24. Pezzati R, Bossi M, Podini P, Meldo-
nesi J, Grohovaz F. High resolution
calcium mapping of the endoplasmic reti-
culum-Golgi-exocytic membrane system.
Mol Biol Cell 1997: 8: 15011512.
25. Porter AE, Patel N, Skepper JN, Best
SM, Bonfield W. Effect of sintered sili-
cate-substituted hydroxyapatite on remo-
deling processes at the bone-implant
interface. Biomaterials 2004: 25: 3303
3314.
26. Ward BC, Webster TJ. The effect of
nanotopography on calcium and phos-
phorus deposition on metallic materials
in vitro. Biomaterials 2006: 27: 3064
3074.
27. Wroblewski R, Wroblewski J, Lund-
strom H, Edstrom L, Jansson E. Energy
dispersive X-ray microanalysis, neutron
activation analysis and atomic absorption
spectroscopy - comparison using biologi-
cal specimens. Scanning Microsc 1989: 3:
467.
28. Xue W, Liu X, Zheng XB, Ding C. In
vivo evaluation of plasma-sprayed coat-
ing after alkali modification. Biomaterials
2005: 26: 30293037.
29. Xue W, Liu X, Zheng XB, Ding C. In
vivo evaluation of plasma-sprayed wol-
lastonite coating. Biomaterials 2005: 26:
34553460.
Address:
Inigo Braceras
Mikeletegi Pasealekua 2
Teknologi Parkea
E-20009 Donostia-San Sebastian
Spain
Tel: +34 943 003700
Fax: +34 943 003800
E-mail: ibracera@inasmet.es