Biomaterials 76 (2016) 313e320

Contents lists available at ScienceDirect

Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials

In vitro and in vivo evaluation of a new zirconia/niobium biocermet

for hard tissue replacement

a, *, J.S. Moya a, c, R. Couceiro b, C.F. Gutie

rrez-Gonza

lez c, F. Guitia

n d,
J.F. Bartolome
A. Martinez-Insua d
a
Instituto de Ciencia de Materiales de Madrid (ICMM), Consejo Superior de Investigaciones Cientcas (CSIC), Sor Juana In

es de la Cruz 3, 28049 Madrid,
Spain
b
Translational Medical Oncology, Health Research Institute of Santiago (IDIS), Fundacion Ramon Dominguez, SERGAS, 15706 Santiago de Compostela, Spain
c

n en Nanomateriales y Nanotecnologa (CINN), Consejo Superior de Investigaciones Cientcas (CSIC), Universidad de Oviedo (UO),
Centro de Investigacio
Principado de Asturias (PA), Avenida de la Vega 4-6, 33940 El Entrego, Asturias, Spain
d

mica de Galicia, Universidad de Santiago de Compostela (USC), Avda. Maestro Mateo, s/n. Campus Vida, 15706 Santiago de Compostela,
Instituto de Cera
Spain

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 16 April 2015
Received in revised form
19 October 2015
Accepted 26 October 2015
Available online 27 October 2015

Metals and ceramics are commonly used in orthopaedics, dentistry and other load bearing applications.
However, the use of ceramic matrix composites reinforced with biocompatible metals for heavy loadbearing hard tissue replacement applications has not previously been reported. In order to improve
the reliability and the mechanical properties of biomedical implants, new zirconiaeNb composites have
been recently developed. The aim of this study was to investigate the biological tolerance of these new
zirconia/Nb biocermets implants with both in vitro and in vivo approaches. At rst, human bone marrow
derived mesenchymal stem cells were cultured on sintered biocermet discs with polished surfaces and
were compared with responses to niobium metal. In vitro, the biocermets showed no deleterious effect
on cell proliferation, extra-cellular matrix production or on cell morphology. Furthermore, the biocermet
showed a higher percentage of cell proliferation than Nb metal. On the other hand, the bone response to
these new zirconia/Nb biocermets was studied. Cylinders of biocermets, as well as commercially Nb rod
were implanted in the tibiae of New Zealand white rabbits. All the animals were euthanatized after 6
months. The specimens were processed to obtain thin ground sections. The slides were observed in
normal transmitted light microscope. A newly formed bone was observed in close contact with material
surfaces. No inamed or multinucleated cells were present. This study concluded that zirconia/Nb
composites are biocompatible and osteoconductive. The ceramic-metal composite has even better
osteointegration ability than pure Nb. In conclusion, zirconiaeNb biocermet is suitable for heavy loadbearing hard tissue replacement from the point of view of both mechanical properties and
biocompatibility.
2015 Elsevier Ltd. All rights reserved.

Keywords:
Zirconia
Niobium
Ceramic-metal composites
Implants
Bone growth

1. Introduction
Among different kinds of biomaterials, metals and metallic alloys
are being used in orthopaedics, dentistry and other load bearing
applications mainly due to their mechanical properties. Ceramics
are also being pushed in this sector as a consequence of their
chemically inert nature, hardness and even their high bioactivity.
Broadly, all structural biomaterials are being developed to maintain

* Corresponding author.

).
E-mail address: jbartolo@icmm.csic.es (J.F. Bartolome
http://dx.doi.org/10.1016/j.biomaterials.2015.10.058
0142-9612/ 2015 Elsevier Ltd. All rights reserved.

a balance between the mechanical properties of the replaced tissues

and the biochemical possible effects that materials may induce on
them. Both areas are of great importance because of their inuence
on the nal clinical success of this kind of materials. Most (if not all)
implants integrated in biological systems, could fail in any aspect.
Ceramic implants present low toughness and susceptibility to aws,
when introduced either during processing or in service. Furthermore, zirconia (3Y-TZP) based ceramics when exposed to a lowtemperature range for a long period in air or water becomes
instable and experiences detrimental strength degradation due to
the tetragonalemonoclinic phase transformation; this is the so-

314

J.F. Bartolom
e et al. / Biomaterials 76 (2016) 313e320

called low-temperature degradation (LTD), which is also known as

aging [1]. Elastic properties (Young Modulus) similarity of the
biomaterial to the host or replaced tissue is another important issue.
If a stiff ceramic implant is placed in the bone, the bone will be
subjected to lower mechanical stresses, and consequently bone will
resorb leading aseptic loosening of the prosthesis. Metals implants
are released by various mechanisms, including corrosion and mechanically accelerated electrochemical processes such as stress
corrosion and corrosion fatigue when immersed in body uids.
Corrosion products can accumulate in tissues encapsulating the
implant [2]. Cobalt and chromium ions can be detected throughout
the body in patients who have hard metal on-metal hip replacements and their possible negative effects are not yet
completely known. Recent publications [3,4] express large concerns
over hypersensitivity reactions and possible carcinogenic (cancer
causing) effects in the medium-short time. It has been reported that
of the 70 metals in the periodic table, only Zr, Ti, Nb and Ta support
broblast growth and osteointegration [5]. Magnetic nature of
stainless steel and cobaltechromium implants causes an interaction
with the magnetic elds present during magnetic resonance imaging (MRI) resulting in device movement, heating, development of
an artefact or distortion on the collected image. This artefact problem is more critical on spinal implants. Along with its good radioopacity, niobium exhibits minor MRI compatibility (bulk magnetic
susceptibility: Stainless steel 3520e6700  106, Co 250,
Ni 600, Fe 200.000, Zr 109  106, Nb 237  106) [6].
Therefore, a specic clinical need can only be fullled by
designing materials, with an optimal combination of properties.
Ceramic matrix materials reinforced with biocompatible metals
(biocermets) can favorably combine the dissimilar properties of
ceramic and metal components in one material with superior
properties when compared with their pure counterparts. Research
in this eld is scarce, only a few papers can be found in the literature investigating both the mechanical and biological properties of
materials like hydroxiapatite, bioactive glass or wollastonite matrix
reinforced with Ti, stainless steel, and Ag particles [7e15].
There are many reports about in vitro or in vivo biocompatibility
on monolithic zirconia ceramics [16e18]. Efcacy of osteointegration of niobium metal systems has been demonstrated through
animal experimentation [19,20]. However, there is a lack of studies
that evaluate osteointegration of zirconia-niobium composite
(biocermets) implants retrieved after a period of implantation in
animals. Composite implants used in this study were fabricated as
an attempt not only to improve the mechanical properties but also
to combine the advantages of the osteoconductive Nb and the
biocompatibility of ZrO2. The development of technologies for the
production of new biocompatible materials has been motivated by
the increasing demand for materials capable to support new
specications and applications. The biocompatible character of
biocermets, alongside with their mechanical and tribological
properties, bridge the gap between biofunctional and structural
materials. Previous studies have pointed out that this new 3Y-TZP/
Nb composite exhibits an optimal combination of aging free,
crack growth resistance (toughness value of 16 MPa m1/2, with a
reported bending strength (sf) of 800 MPa) and tribological characteristics never reached before [21e27]. Moreover, it is possible to
tailor the elastic properties of the biocermets controlling the fraction/shape of metallic second phase. In this particular case (3Y-TP/
20vol% Nb) the modulus of elasticity is 135 GPa, close to titanium
Alloys-Ti6Al4V Grade 5 (z120 GPa).
The purpose of this investigation is to evaluate the in vitro human bone marrow derived mesenchymal stem cells proliferation
response of polished discs, and to investigate bone tissue reactions
around cylindrical implants made of commercially pure niobium as
a reference material and biocermet. The null hypothesis tested was

that zirconia/Nb composites are biocompatible and osteoconductive and they have similar osteointegration ability than
pure Nb.
2. Materials and methods
Details on the starting ceramic and metallic powders and the
processing of the composites have been described previously [21].
The powders were consolidated into composite specimens by hot
press. The hot pressing temperature was 1400  C during 1 h with
heating and cooling rates of 600  C/h in an inert 100% Ar atmosphere. On reaching the hot pressing maximum temperature, a
uniaxial pressure of 45 MPa was applied. Both pressure and temperature were held for 1 h. As result, discs with 20 mm diameter
and 12 mm thickness were obtained. Cylindrical bars (5 mm
length  2 mm diameter) and discs (20 mm diameter  2 mm
thickness) were cut and machined from the pieces. Additionally,
disks (20 mm diameter  2 mm thickness) and cylinders (5 mm
length  2 mm diameter) were saw cut from a rod of 20 mm
diameter and 50 mm length (Goodfellow, Nb007940, 99.9% purity)
and from a rod of 2 mm diameter and 100 mm length (Goodfellow,
Nb007910, 99.9% purity) respectively.
2.1. Microstructure and surface characterization
The microstructure of sintered specimens was studied on
surfaces polished down to 1 mm by scanning electron microscopy
(SEM, Phenom G2). Roughness measurements of cylinders were
carried out by using SEM with the 3D Roughness Reconstruction
application (Phenom TM Pro Suite). The cut-off lter was 0.03 mm
and the evaluation length was 500 mm per measurement. Three
samples of each surface type were scanned to evaluate the
average surface roughness (Ra) of the surfaces at ten different
locations.
2.2. In vitro study
2.2.1. Human mesenchymal cell isolation and culture
To investigate the cellular responses to the commercially pure
niobium and developed biocermet, mesenchymal stem cells
derived from human bone marrow were used (hBMSCs). hBMSCs
were obtained by the Department of Orthopaedic Surgery (University of Santiago de Compostela Clinical Hospital) during routinary hip prosthesis surgeries by ethically approved protocols. Bone
marrow was stored in heparin coated tubes and taken to the cell
culture laboratory to proceed with mesenchymal stem cell isolation. A Ficoll protocol as described by Yeo et al. [28] was used.
Briey, cells are separated through a density gradient where an
intermediate band is formed after a 2000 rpm centrifugation. This
band is washed with DMEM (10%, Strep, Gibco) centrifuged for a
second time and cultured in 75 cm2 asks (Corning). Cultures are
kept at a temperature of 37  C and 5% CO2 in an incubator until a
90% conuence is reached. Cells are tripsinised and subcultured on
the surface of the assayed materials at a density of 60  103 cells/
cm2. Cultured materials are assayed for different parameters after 2,
7 and 10 days.
2.2.2. Biocompatibility tests
2.2.2.1. Cell proliferation by MTT assays. The survival and proliferation of hBMSCs on pure niobium and biocermet discs was examined with a MTT assay after 2, 7 and 10 days of culture. The MTT
assays were carried out as per manufacturer's protocol
(SigmaeAldrich).
Briey, hBMSCs were seeded on the sample discs at a density of
25  103 cells per well in a 24-well plate. At each time point, the

J.F. Bartolom
e et al. / Biomaterials 76 (2016) 313e320

medium were removed, MTT solution was added, and cells were
incubated overnight. After removal of the MTT solution, the purple
formazan crystals were dissolved in 100 mL of dimethyl sulphoxide
(DMSO) by shaking the plate for 15 min, and 50 mL solution of each
well was added into a new 24-well plate. The OD value was
quantied by measuring the absorption at 570 nm using an automated plate reader (PerkinElmer).
2.2.2.2. Apoptosis: caspase activity assay. Caspase-3 activity was
measured in supernatants by a Quantikine immunoassay (ELISA)
active caspase-3 kit (R&D Systems, Minneapolis, MN). Briey,
cytoplasmic protein extract was incubated with biotin-ZVKDuoromethylketone inhibitor, which covalently binds and makes
detectable the active caspase-3. A monoclonal antibody specic for
active caspase-3 was precoated onto a microplate, and active protease concentrations were assessed in cell lysates, according to the
manufacturer's instructions.
2.2.2.3. Cell morphology. Cell morphology was assessed under
scanning electron microscopy (SEM, Phenom G2). Human bone
marrow derived mesenchymal stem cells were cultured on metallic
Nb and biocermet substrates (DMEM FCS 20% Gent) at a
density of 250  103 cell/cm2. Cultures were kept in a CO2 incubator
for 48 h (5% CO2, 37  C). After this period, culture media was
removed and cells were washed twice with PBS. Immediately after
the last wash, a xing solution (1% Glutaraldehyde, 1% Paraformaldehyde) was added to the culture wells and kept at 25  C for
48 h. Once cells were xed, a dehydration process took place by
means of serial dilutions of EtOH.
2.2.2.4. Confocal microscopy. Live/dead staining. Calcein-AM and
propidium iodide were used to stain the hBMSCs cultured on the
glass ceramic pellets. After 48 h of incubation the samples were
gently washed with D-MEM. The viable and non viable cells were
visualized by using a confocal laser scanning microscope (CLSMSP2, Leica), the viable cells appear uorescent green, whereas
nonviable cells appear uorescent red.
2.2.2.5. Statistical analysis. Mintab 16 (Minitab INC) was used for
the statistical data analysis. T-student tests were carried out with
an a 0.05.
2.3. In vivo study
2.3.1. Animals and surgical technique
Five adult New Zealand white rabbits (Charlie Rivel), aged from
9 to 10 months (3.5 Kg), were used for the experiments. All surgical
procedures were done under strict aseptic protocol. The animals
were premedicated with diacepam 0.2 ml/kg (Valium 10, Roche),
ne 1000, Merial,
and anaesthetized with ketamine 10 mg/kg (Imalge
France) and medetomidina (0.1 mg/kg) (Domtor, Orion. Finland)
injected intramuscularly in the hind leg. The animal tibialis anterior
face was shaved. The skin was disinfected with a povidone-iodine
solution 10% (Betadine, Medapharma). Inltrative anesthesia
(Ultracain 40/0.005, Normon, 1.8 ml) was employed injected subcutaneously in the anterior tibialis face. Four cylindrical bars with a
length of 5 mm and diameter of 2 mm, were placed in each animal,
two in each medial proximal tibia. Surgical preparations for the
cylinders were done using rst a pilot drill (Precision drill 33071
Nobel Biocare), and then a 2 mm twist drill (Twist Drill 32296.
Nobel Biocare). Careful drilling was done with a low rotary hand
piece (KaVo Intrasurg 300) never exceeding 2000 rpm and under
profuse saline cooling. Metallic bars were placed on the right tibia,
while biocermets were placed on the left tibia. All cylinders were
more than 10 mm apart. The cylinders were impacted and the

315

surface end was placed oss with bone. The periostium was sutured
with continuous monolament reasorbable suture (3-0 Monocryl
Ethicon). The skin was sutured with interrupted threaded sutures
(Vicryl 3-0, Ethicon). Skin was again soaked with povidone solution.
After 6 months, rabbits were euthanized with a lethal dose of ketamine/diacepam injection.
2.3.2. Histological preparation and examination
After euthanized the entire tibias were removed. The position of
the implants was located via radiologic examinations. The tibiae
were xed in buffered formalin (10%) for one week. The size of each
sample was reduced by separating the two implants of each tibia.
Following Donath and Breuner method [29], dehydration of the
samples was achieved by immersion in increasing concentration of
alcohol solutions. The samples were inltrated with Technovit
7200 resin (Kulzer). After photopolymerization of the inltrated
blocks, thin ground slides were obtained. The preparations were
dyed with Harris Hematoxiline (Papanicolau, Merck, Germany) and
Wheatley's modication of thrichromic stain (Chromotrope 2R,
Newcommersupply, USA) and preserved with Canada balsam solution (Fluka Biochemika, USA). Preparations were examined by
using a transmitted light microscope (Optiphot, Nikon, Japan)
equipped with a digital Camera (DP-12, Olympus, Japan). The
analysis of osteocyte lacunar density was performed in
100  100 mm reticle at 10 different areas of old and newly formed
bone.
2.3.2.1. Histomorphometric evaluation. Histomorphometry was
done employing an Olympus SZX12 microscope equipped with a
Olympus DP12 camera. Images obtained were processed with image J-1.46r software (NIH, USA) to measure the bone-to-implant
contact (BIC) ratio, dened as the proportion of bone in direct
contact with the implant surface. When necessary, polarized light
microscope was employed to determine the boundaries of the
newly formed bone. All measurements were taken by the same
investigator and boundaries were revised by a different one. To
determine the reproducibility and the measurement error, ve
randomly selected slides where measured three times, in three
different days [30].
2.3.2.2. Statistical analysis. Given the relatively small size of the
sample (60 histological slides), the normality of the histomorphometry data was tested with a KeS test and then a t-test was
employed to compare the means. The statistical package employed
was the IBM-SPSS 18.0 (Chicago, IL). The level of signicance was
set at P  0.05.
3. Results and discussion
3.1. Microstructure and roughness
SEM micrograph of ZrO2eNb composite is shown in Fig. 1. In this
micrograph the darker and bright phases are zirconia and Nb
grains, respectively. The Nb particles are uniformly dispersed in the
matrix and no porosity is observed.
The machined process resulted in a similar value between discs
and cylinders of different materials but substantially rougher surface on the cylinders compared with the polished disc surface
(mean surface roughness values (Ra): 0.04 0.01 and
0.09 mm 0.01 for Nb and biocermet dics vs 1.06 0.2 and
0.78 mm 0.1 for Nb and biocermet cylinders respectively).
3.2. In vitro cytocompatibility of discs
Mesenchyamal cells were chosen in this experiment as it was

316

J.F. Bartolom
e et al. / Biomaterials 76 (2016) 313e320

Fig. 1. Electron micrograph of ZrO2eNb composite polished disk. The darker and
bright phases are zirconia and Nb grains, respectively.

considered as an important approach for testing the new materials.

It is known that a primary cell will not have its cytoskeleton or
proliferation patterns modied if compared to cell lines such as
SAOS-2 [31]. It is important to note that these cells could also be
used for further studies such as in vitro osteoinduction.
Proliferation of mesenchymal stem cells to substrates is show in
Fig. 2. Cells were plated onto each of the substrates and incubated
on the various substrates for 2 days, 7 days or 10 days. MTT assay
results are presented as mean standard deviation. Fig. 2 shows
how there is an increase in cell proliferation for both materials.
There is however a larger proliferation rate after 10 days of culture
for the ZrO2eNb samples, showing a material more prone for cell
colonization.
Fig. 3 shows a steady increase of Caspase 3 activity in both
samples, this is due to the intrinsic apoptosis rate in the culture.
However, Nb shows a somehow larger apoptosis rate for the long
term group. Caspase 3 activity is low in both materials proving the
biocompatibility of Nb and the newly described biocermet.
Fig. 4 shows the SEM study of cell morphology after 48 h of
incubation. Cell spreading and proliferation is observed clearly in
all the samples (Fig. 4A and B). Almost the whole surface is covered
with cells at this stage, and a large amount of extracellular matrix
(ECM) is now visible (Fig. 4C). Human Mesenchymal cells have

Fig. 2. MTT assay of cells on Nb and ZrO2eNb substrates after 2, 7 and 10 days of
culture. T-test results each time point, n 10 and a 0.05, H0: m1 m2; >H1: m1>m2 Pvalues were all as follow: 2 days 0.04, 7 days 0.02, 10 days 0.02. All values were
bellow 0.05, hence H0 was rejected and H1 accepted.

Fig. 3. Caspase assay results for ZrO2eNb and Nb. T-test results each time point, n 10
and a 0.05, H0: m1 m2; >H1: m1>m2. P-values were all as follow: 2 days 0.06, 7
days 0.03, 10 days 0.02. P-Value for 2 days is above alpha, leading to the acceptance
of H0, thus assuming no signicant differences between groups. However the rest of
the time points are below such level.

already undergone through an attachment process and spreading is

clearly observed. Slender lopodia crosses the surface looking forward to link with other surrounding cells, promoting cellecell
communications and ECM deposition (Fig. 4D). Cells morphology
was not obviously different when adherent to any of the materials
and the surface roughness did not markedly inuence cell adherence or shape. This might have been expected because all the
surfaces were relatively smooth whether polished.
Cell proliferation and viability is optimal for both surfaces when
observed in a confocal microscope (Fig. 5). After 10 days of culture,
the biocermet sample appears to show a greater concentration of
cells on the surface and even some alignment patterns. The Nb
sample on the other hand is not as colonized, supporting the observations from the ELISA essays.
The results of this study indicate that ZrO2eNb biocermet is a
suitable material for human mesenchymal stem cells growth and
differentiated function. This is supported by the initial cell attachment to biocermet, and cell morphology on biocermet, not being
different from metallic Nb. In addition, lower apoptosis and higher
proliferation values of cells on biocermet compared to the Nb
tested, was observed.
3.3. In vivo biocompatibility
The growing status of newborn bones in defective bone region
around the implants and the interfacial morphology between the
implant and newborn bone was observed by light microscopic as
shown in Figs. 6 and 7 for biocermet and Nb metal respectively.
Histological observations after 6 months of the implantation
show that some immature newborn osteoids could be found in NB
regions for both composite and pure Nb metal as shown in Figs. 6A
and 7A. These new bone tissues can contact directly with the Nb
metal and composite and no brous tissue was observed at the
interface between the implants and new bones. No inammatory
response such as the presence of foreign-body giant cells and
inltration of inammatory cells were observed in the histological
examination of these implants. It is also indicated that nor the
ceramic nor the metal released toxic particles or products responsible for a cell death or apoptosis in the site of implantation.
The most relevant feature is the complete stabilization of the
biocermet cylinder in the tibial diaphysis (Fig. 6). There is a close
apposition of bone on the surface of the implant in two places:
where the implant passes through the compact bone in the facies
medialis tibiae and in the apical end where the implant is

J.F. Bartolom
e et al. / Biomaterials 76 (2016) 313e320

317

Fig. 4. Scanning electron micrographs of mesenchymal stem cells plated onto the A) niobium, B) C) and D) biocermet substrates. Microphotographs are of typical elds.

Fig. 5. CLSM image of the hBMSCs grown on: A) Nb surface and B) ZrO2eNb surface during culture for 10 days. hBMSCs were stained with green uorescent calceim-AM. (For
interpretation of the references to color in this gure legend, the reader is referred to the web version of this article.)

surrounded by a close net of trabeculae newly formed from the

endostium. The intermediate portion of the implant is in close
contact with the bone marrow. The most supercial end of the
implant is partially covered by new bone formed from the bone
surface periostium. The apical end of the implanted cylinder shows
also osteoconductive properties because some trabecaulae formed
from the endostium grows and migrates over the implant surface.
This newly formed bone is strongly vascularized, vessels are in
close relation with the implant surface. New bone has lamellar
structure and in some places Haversian formations can be seen. In
any place brous or connective tissue reaction can be observed in
the interface. Bone grows in close contact with the implant surface.
The osteocite lacunae are regularly occupied by osteocites cells,
and the newly formed bone had an osteocite lacunae density of

26 0.5/100m2. These lacunae are disposed following a mature

bone typical structure (lamellar or haversian bone) but there are
some remnants of woven bone areas with less mineralization.
Some reversal lines can be seen showing remodeling activity inside
the bone. The lacunae density in the old bone was 23.6 0.5/100m2.
Differences were statistically signicant for a P 0.01. Inammatory inltration as well as dysplasia or atypical cellular forms could
not be seen in any part of the preparations.
The niobium rod surface is covered by the diaphysal bone
(Fig. 7). The newly formed bone covers the surface end and the
apical end of the rod, and mainly presents a mature structure. In the
cavitas medullaris the bone grows over the metal surface in the
boundaries of the endostal surface. The close contact with bone is
formed by mature bone with Haversian structures and by a small

318

J.F. Bartolom
e et al. / Biomaterials 76 (2016) 313e320

Fig. 6. A) Apical end of the biocermet implant (I). Apical zone of the implant surrounded by new bone (NB) formed from diaphysis endostal surface. Note the intense vascularization
of the new bone coming from the adjacent diaphysal old bone (OB). Note the different orientation of the osteocite lacunae. B) Interface: biocermet implant (I) and diaphysal bone
(HB). Note the close contact between bone and implant. Note the Haversian structure of the compact bone (HB). C) Bone-biocermet implant interface. Lesser mineralized bone
shows the pinkepurple color, and is in close contact with implant surface (I) and surrounding the vessels (V) and marrow spaces (BM). Note the Haversian structure of the
remodeled bone. D) Bone biocermet implant-interface in the boundaries with bone marrow diaphysal space (BM): new bone with lamellar structure (NB) in close contact with
implant surface (I), note the vascular structures (V) passing in the nearby of the implant. Old bone (OB) with half-Haversian structure, note the osteocites embedded in the lacunae.
(For interpretation of the references to color in this gure legend, the reader is referred to the web version of this article.)

Fig. 7. A) Apical end of the Niobium rod. The new bone (NB) grows on the implant (I) surface, surrounding the end and marrow spaces (BM). Note the compact old bone (OB) and
the bone marrow entrapped between the newly formed structures (NB). B) Surface of the Niobium rod in the entrance of the cavitas medullaris tibiae. (CM). Observe the supplementary new bone (NB) formed from the old bone (OB) diaphysis endostal surface (arrows). C) Close up of the bone-implant interface. The new bone (NB) is formed in close
contact with the implant surface (I). Observe the thin strip of bone with lower mineralization (pinkepurple) in contact with implant (I). Note the remodeling Haversian structures
(HV) delimited by the cemental line (arrows). D) Close up of the implant (I) apical end. Note mature Haversian bone in close contact with implant (HV). Vascular spaces (V), some of
them, in close contact with niobium surface. Note the thin strip of immature bone in close contact with implant (pinkepurple) (S). (For interpretation of the references to color in
this gure legend, the reader is referred to the web version of this article.)

J.F. Bartolom
e et al. / Biomaterials 76 (2016) 313e320

and thin discontinuous strip of newly formed bone, which presents

a lower mineralization. The lacunae are occupied by osteocites
disposed mainly in Haversian structures, delimited by cemental
lines. The osteocite density is 23.1 1/100 mm2. Bone marrow is
mature and normal. There is a lack of inammation, brous
encapsulation or dysplasia.
Histological observations after six months of the implantation
indicate the newly formed bone in both kinds of implants covering
the majority of the rod surface, and mainly present a mature
structure as shown in Figs. 6 and 7, which suggests that the
biocompatibility and osteoconductivity of the biocermet may be
very good like those of pure Nb. After 6 months the amount of bone
and bone-to-implant contact area was signicantly higher for the
biocermet compared to the niobium metal rod. The mean contact
index (BCI) presented the following values: Biocermet: 68.5 5.1
and Niobium rod: 50.3 4.3, with a signicant difference between
the groups for p 0.02. No brous tissues came into being at the
interface between the implants and new bones during the period
after the implants were xed into the hard tissues. On the other
hand, it could be found that both materials were fully osteointegrated with newborn bone tissues. From the point of view of
osteointegration ability of the implants with newborn bone tissues
after six months in vivo, biocermet with excellent biocompatibility
has a better afnity to body tissues compared with pure Nb. This
may be due to the coexistence of ceramic and metal grains and the
microstructure of the composite. Our results suggest that the
ZrO2eNb biocermet synergistically enhanced osteointegration. It
has been reported that the formation of NbeOH on niobium contributes to its bioactivity [32,33]. In air or any aqueous electrolyte,
an oxide is spontaneously formed on the metal. This surface oxide
is hydroxylated and has amphoteric, or bipolar character. The
chemisorption ability of such surfaces is well known [34]. In
particular, peptides and amino acids act as ligands and attach to Nb
oxide by replacement of the surface hydroxyl. The terminal
carboxyl and amino groups of amino acids and proteins bind with
the surface hydroxyls. In previous work [23], we have observed a
direct contact between ceramic and metallic grains at the biocermet interfaces without any additional phases. A solid solution of
Nb2O5 in ZrO2 takes place [21,35,36] and it may well be that the
oxygen is dissolved and distributed statistically in the niobium [37].
The ceramic-metal bonds produce highly reactive polar oxide surfaces. Therefore, ZrO2eNb interfaces possess high activity to
generate NbeOH groups. These appear to be the main reason for
the excellent osteogenic activity observed from biocermet but more
studies are needed to elucidate the accurate mechanism.
In summary, the in vitro and in vivo studies indicate that
ZrO2eNb composite is fully biocompatible and can integrate with
bone. The cell proliferation (%) and bone-implant interface around
biocermet implants are higher (68.5% vs 50.3% for Nb metal) to that
observed for Nb implants. The surfaces of the biocermet implants
appeared to be highly biocompatible, and no gaps, brous tissue,
multinucleated cells, or inammatory cell inltrate were found at
the bone-implant interface. It is important to point out that the
in vivo an in vitro studies show a similar trend, taking into account
the signicantly different mean surface roughness values (Ra) of
cylinders (0.98 mm 0.1) compared with the polished disc surface
(0.09 mm 0.01). The results obtained reject the null hypothesis.
4. Conclusions
The following conclusions can be drawn:
- ZrO2eNb biocermet shows the same cell morphology and
viability when compared to Nb metal material. However, in
accordance with the presented apoptosis and proliferation

319

values, ZrO2eNb composite is an optimal material for cell proliferation. All the samples were found to be biocompatible,
however the biocermet creates a more cell friendly surface.
- Observation during in vivo study has conrmed effectiveness of
osseointegration within this period of time. New bone was
observed around system of the on rod devices at retrieval date.
Bone in growth however, proved to be slower and of less intense
degree in the case of pure metal than that resulting in the biocermet. ZrO2eNb composite has better osteoconduction and
osseointegration abilities than Nb metal after 6 months in vivo.
These results for the in vivo studies are largely consistent with
the in vitro studies.
- The results obtained in the present investigation indicate that
biocermets are suitable for orthopedic and dental applications
and have large clinical potential.
Acknowledgments
This work was supported by the Spanish Ministry of Science and
Innovation (MICINN) under the project MAT2012-38645.
References
[1] J. Chevalier, L. Gremillard, S. Deville, Low-temperature degradation of zirconia
and implications for biomedical implants, Annu. Rev. Mater. Res. 37 (2007) 1.
[2] J.J. Jacobs, J.L. Gilbert, R.M. Urban, Current concepts review: corrosion of metal
orthopaedic implants, J. Bone Jt. Surg. 80A (1998) 268e282.
[3] H. Pandit, S. Glyn-Jones, P. McLardy-Smith, R. Gundle, D. Whitwell,
C.L.M. Gibbons, et al., Pseudotumours associated with metal-on-metal hip
resurfacings, J. Bone Jt. Surg. Br. 90 (7) (2008) 847e851.
[4] Y.-M. Kwon, S.J. Ostlere, P. McLardy-Smith, N.A. Athanasou, H.S. Gill,
D.W. Murray, Asymptomatic Pseudotumors after metal-on-metal hip
resurfacing arthroplasty. Prevalence and metal ion study, J. Arthroplasty 26
(2011) 511e514.
[5] Y. Okazaki, E. Gotoh, Corrosion resistance, mechanical properties, fatigue
properties, and tissue response of Ti-15Zr-4Nb-4Ta alloy, in: L.D. Zardiackas,
M.J. Kraay, H.L. Freese (Eds.), Titanium, Niobium, Zirconium and Tantalum for
Medical and Surgical Applications, ASTM STP 1471, ASTM International, West
Conshohocken, PA, 2006, p. 120.
[6] B. O'Brien, J. Stinson, W. Carroll, Development of a new niobium-based alloy
for vascular stent applications, J. Mech. Behav. Biomed. Mater. 1 (2008) 303.
[7] C.Q. Ning, Y. Zhou, In vitro bioactivity of a biocomposite fabricated from HA
and Ti powders by powder metallurgy method, Biomaterials 23 (14) (2002)
2909e2915.
[8] A. Arin, A.B. Sulong, N. Muhamad, J. Syarif, M.I. Ramli, Material processing of
hydroxyapatite and titanium alloy (HA/Ti) composite as implant materials
using powder metallurgy: a review, Mater. Des. 55 (2014) 165e175.
[9] E. Verne, E. Bona, A. Bellosi, C. Vitale Brovarone, P. Appendino, Na2O-CaO-SiO2
glass-ceramic matrix biocomposites, J. Mater. Sci. 36 (11) (2001) 2801e2807.
[10] D.K. Pattanayak, R.C. Prasad, B.T. Rao, T.R. Rama Mohan, Apatite wollastonitetitanium biocomposites: synthesis and in vitro evaluation, J. Am. Ceram. Soc.
89 (7) (2006) 2172e2176.
[11] C.L. Chu, X.Y. Xue, J.C. Zhu, In vivo study on biocompatibility and bonding
strength of hydroxyapatite-20vol%Ti composite with bone tissues in the
rabbit, Biomed. Mater. Eng. 16 (3) (2006) 203e213.
[12] E. Claxton, B.A. Taylor, R.D. Rawlings, Processing and properties of a bioactive
glass-ceramic reinforced with ductile silver particles, J. Mater. Sci. 37 (2002)
3725e3732.
[13] X. Fan, J. Chen, J.-P. Zou, Q. Wan, Z.-C. Zhou, J.-M. Ruan, Bone-like apatite
formation on HA/316L stainless steel composite surface in simulated body
uid, Trans. Nonferrous Met. Soc. China 19 (2009) 347e352.
[14] S. Nath, S. Kalmodia, B. Basu, Densication, phase stability and in vitro
biocompatibility property of hydroxyapatite-10 wt% silver composites,
J. Mater. Sci. Mater. Med. 21 (2010) 1273e1287.
[15] Q. Chang, D.L. Chen, H.Q. Ru, X.Y. Yue, L. Yu, C.P. Zhang, Toughening mechanisms in iron-containing hydroxyapatite/titanium composites, Biomaterials
31 (2010) 1493e1501.
[16] C. Piconi, G. Maccauro, Zirconia as a ceramic biomaterial, Biomaterials 20
(1999) 1e25.
[17] Michael Hisbergues, Sophie Vendeville, Philippe Vendeville, Review zirconia:
established facts and perspectives for a biomaterial in dental implantology,
J. Biomed. Mater. Res. Part B Appl. Biomater. 88B (2009) 519e529.
ller, H. Terheyden, Y. Ail, N.M. Purcz, K. Hertrampf, A. Tabakov,
[18] B. Mo
E. Behrens, J. Wiltfang, A comparison of biocompatibility and osseointegration
of ceramic and titanium implants: an in vivo and in vitro study, Int. J. Oral
Maxillofac. Surg. 41 (2012) 638e645.
[19] C.B. Johansson, T. Albrektsson, A removal torque and histomorphometric
study of commercially pure niobium and titanium implants in rabbit bone,

320

J.F. Bartolom
e et al. / Biomaterials 76 (2016) 313e320

Clin. Oral Implants Res. 21 (1991) 24e29.

[20] H. Matsuno, A. Yokoyama, F. Watari, M. Uo, T. Kawasaki, Biocompatibility and
osteogenesis of refractory metal implants, titanium, hafnium, niobium,
tantalum and rhenium, Biomaterials 22 (2001) 1253e1262.

, C.F. Gutie

rrez-Gonz


n, J.S. Moya, Synergisitc
[21] J.F. Bartolome
alez, C. Pecharroma
toughening mechanism in 3Y-TZP/Nb composites, Acta Mater. 55 (17) (2007)
5924e5933.

, A. Ferna

ndez, S. Lopez[22] R. Torrecillas, J.S. Moya, L.A. Daz, J.F. Bartolome
Esteban, Nanotechnology in joint replacement, Wiley Interdiscip. Rev. Nanomedicine 1 (5) (2009) 540e552.

, J.I. Beltra

n, C.F. Gutie

rrez-Gonz


n,
[23] J.F. Bartolome
alez, C. Pecharroma
~ oz, J.S. Moya, Inuence of ceramic/metal interface adhesion on crack
M.C. Mun
growth resistance of zirconia/Nb ceramic matrix composites, Acta Mater. 56
(2008) 3358e3366.

rrez-Gonza

lez, J.S. Moya, F.J. Palomares, J.F. Bartolome

, Low-tem[24] C.F. Gutie
perature aging degradation-free 3Y-TZP/Nb composites, J. Am. Ceram. Soc. 93
(7) (2010) 1842e1844.

rrez-Gonza

lez, A. Smirnov, J.F. Bartolome

, Aging effect on the
[25] C.F. Gutie
tribological behavior of a novel 3Y-TZP/Nb biocomposite against Ultra High
Molecular Weight Polyethylene (UHMWPE), J. Am. Ceram. Soc. 95 (3) (2012)
851e854.

, J.S. Moya, Mechanical and tribological
[26] T. Rodrguez-Suarez, J.F. Bartolome
properties of ceramic/metal composites: a review of phenomena spanning
from the nanometer to the micrometer length scale, J. Eur. Ceram. Soc. 32 (15)
(2012) 3887e3898.

rrez-Gonza

lez, J.F. Bartolome

, Cyclic fatigue life- and
[27] A. Smirnov, C.F. Gutie
crack-growth behavior of zirconiaeniobium composites, J. Am. Ceram. Soc. 96
(6) (2013) 1709e1712.

[28] C. Yeo, et al., Ficoll-Paque versus Lymphoprep: a comparative study of two

density gradient media for therapeutic bone marrow mononuclear cell
preparations, Regen. Med. 4 (2009) 689e696.
[29] K. Donath, G.A. Breuner, A method for the study of undecalcied bones and
teeth with attached soft tissues. The S
age-Schliff (sawing and grinding)
technique, J. Oral Pathol. 11 (1982) 318e326.
[30] S.M. Yezerinac, S.C. Lougheed, P. Handford, Measurement error and
morphometric studies: statistical power and observer experience, Syst. Biol.
41 (4) (December 1992) 471e482.
[31] Li Ch, Sh Gao, T. Terashita, T. Shimokawa, H. Kawahara, S. Matsuda,
N. Kobayashi, In vitro assays for adhesion and migration of osteoblastic cells
(Saos-2) on titanium surfaces, Cell Tissue Res. 324 (3) (2006) 369e375.
[32] T. Kokubo, H.M. Kim, M. Kawashita, Novel bioactive materials with different
mechanical properties, Biomaterials 24 (2003) 2161e2175.
[33] T. Miyzaki, Development of bioactive materials based on bone-bonding
mechanism on metal oxides, J. Ceram. Soc. Jpn. 116 (2008) 260e264.
[34] S.G. Steinemann, in: G. Ltjering, U. Zwicker, W. Bunk (Eds.), Titanium Science
and Technology, Deutsche Gesellschaft Metallkunde, Oberursel, 1985, pp.
1373e1379.
[35] D.Y. Lee, D.J. Kim, D.H. Cho, Low-temperature phase stability and mechanical
properties of Y2O3- and Nb2O5-co-doped tetragonal zirconia polycrystal ceramics, J. Mater. Sci. Lett. 17 (3) (1998) 185e187.
[36] S. Raghavan, H. Wang, W.D. Porter, R.B. Dinwiddie, M.J. Mayo, Thermal
properties of zirconia co-doped with trivalent and pentavalent oxides, Acta
Mater. 49 (2001) 169e179.
[37] V.P. Kobyakov, V.I. Ponomarev, Specic features of oxygen dissolution in refractory metals in gas-phase deposition, Crystallogr. Rep. 47 (1) (2002)
106e110.