Bioactivity and osteointegration of hydroxyapatite-coated stainless steel and titanium wires used for intramedullary osteosynthesis
Bioactivity and osteointegration of hydroxyapatite-coated stainless steel and titanium wires used for intramedullary osteosynthesis
Arnold V. Popkov 0 1 2 3 4
Elena N. Gorbach 0 1 2 3 4
Natalia A. Kononovich 0 1 2 3 4
Dmitry A. Popkov 0 1 2 3 4
Sergey I. Tverdokhlebov 0 1 2 3 4
Evgeniy V. Shesterikov 0 1 2 3 4
Wire Bone 0 1 2 3 4
0 Natalia A. Kononovich
1 National Research Tomsk Polytechnic University , 30 Lenin Avenue, Tomsk , Russia
2 Head of Research Laboratory for Limb Lengthening and Deformity Correction, Russian Ilizarov Scientific Center for Restorative Traumatology and Orthopaedics , 6 M. Ulianova Street, Kurgan , Russia
3 Laboratory of Morphology, Russian Ilizarov Scientific Center for Restorative Traumatology and Orthopedics , 6 M. Ulianova Street, Kurgan , Russia 640014
4 Laboratory for Limb Lengthening and Deformity Correction, Russian Ilizarov Scientific Center for Restorative Traumatology and Orthopaedics , 6 M. Ulianova Street, Kurgan , Russia
A lot of research was conducted on the use of various biomaterials in orthopedic surgery. Our study investigated the effects of nanostructured calcium-phosphate coating on metallic implants introduced into the bone marrow canal. Stainless steel or titanium 2-mm wires (groups 1 and 2, respectively), and hydroxyapatite-coated stainless steel or titanium wires of the same diameter (groups 3 and 4, respectively) were introduced into the tibial bone marrow canal of 20 dogs (each group = 5 dogs). Hydroxyapatite coating was deposited on the wires with the method of microarc oxidation. Light microscopy to study histological diaphyseal transverse sections, scanning electron microscopy to study the bone marrow area around the implant and an X-ray electron probe analyzer to study the content of calcium and phosphorus were used to investigate bioactivity and osteointegration after a four weeks period. Osteointegration was also assessed by measuring wires' pull-off strength with a sensor dynamometer. Bone formation was observed round the wires in the bone marrow canal in all the groups. Its intensity depended upon the features of wire surfaces and implant materials. Maximum percentage volume of trabecular bone was present in the bone marrow canals of group 4 dogs that corresponded to a mean of 27.1 ? 0.14%, while it was only 6.7% in group 1. The coating in groups 3 and 4 provided better bioactivity and osteointegration. Hydroxyapatite-coated titanium wires showed the highest degree of bone formation around them and greater pull-off strength. Nanostructured hydroxyapatite coating of metallic wires induces an expressed bone formation and provides osteointegration. Hydroxyapatitecoated wires could be used along with external fixation for bone repair enhancement in diaphyseal fractures, management of osteogenesis imperfecta and correction of bone deformities in phosphate diabetes.
Intramedullary osteosynthesis; Hydroxyapatite coating; Osteointegration formation; Osteosynthesis
Biomaterials are a variety of metallic components,
polymers, ceramics or composite materials that are used and/or
adapted for a medical application. A biomaterial may also
be an autograft, allograft or xenograft used as a
transplant material. A lot of research has been conducted to
study the possible use of biomaterials in orthopedics that
would provide bone fixation reinforcement, bone
substitution or be able to induce new bone tissue formation and
osteointegration [1, 2].
In order to improve bioactivity and osteointegration of
metallic implants, their surface is coated with
hydroxyapatite (HA) with the purpose of providing bonding with the
surrounding bone tissue. It has been known that
HA-coated hip implants used in hip arthroplasty showed bioactivity
and osteointegration of their coating with the living bone
tissue . It was shown that primary implant stability is
favored by HA coating which results in an improved
contact between bone and implant. The studies
demonstrated a sufficient osteointegration of HA-coated hip
HA is thought to be an osteoconductive material that
shows inductive ability when implanted extraskeletally .
It was also reported previously that even a
non-soluble metal that contains no calcium or phosphorus can be
an osteoinductive material when treated to form an
appropriate macrostructure and microstructure .
Nailing and plating of a fractured bone are the most
common methods in orthopedic trauma surgery. Recently,
flexible intramedullary nailing with thin wires, HA coated
or uncoated, has been advocated for management of
diaphyseal fractures, lengthening and deformity correction
[6?9]. It was observed that the use of such wires stimulated
bone reparation and reduced the total period of treatment in
clinical settings. Therefore, experimental studies were on
the agenda to unveil these phenomena and obtain their
explanations with fundamental research.
Our experiment was aimed at revealing the processes
that undergo by introduction of HA-coated stainless steel
or titanium wires into the bone marrow canal, and namely
their bioactivity and osteointegration as compared with
regular stainless steel or titanium wires.
Materials and methods
The experiment was conducted on 20 dogs in the age
between one and 3 years. Their mean body mass was
20 ? 2.9 kg.
Four types of wires were used for introduction into the
bone marrow canal in dogs divided into four groups, each
of five dogs. Groups 1 and 2 were dogs with regular
stainless steel or titanium (Ti6Al4 V) wires, respectively
(control groups). HA-coated stainless steel wires or
HAcoated titanium (Ti6Al4 V) wires were introduced into the
tibiae of study groups 3 and 4, respectively. HA coating on
the wires was produced at the research laboratory of Tomsk
Polytechnic University (Tomsk, Russia). The method of
anode microarc oxidation (MAO) in an electrolyte that
contained calcium (Ca) and phosphate (P) compounds was
used to coat the surface of titanium wires (group 4).
Stainless steel wires were first coated by a primary layer of
titanium followed by microarc oxidation for HA coating
(group 3). MAO processing results in mixing the oxidized
substrate with the calcium and phosphate ions supplied
from the electrolyte to form a Ca?P composite.
Microscopic study of wire surfaces was performed using
a scanning electron microscope JSM-840 (JEOL, Japan).
Calcium and phosphate concentrations in the coating were
assessed using an X-ray electron probe analyzer INKA
Energy 200 (Oxford Instruments Analytical, UK).
The operations were performed by one surgical team.
Under general anesthesia, one 2-mm wire with a slightly
bent tip was introduced into the tibia from the medial side
at the level of the tibial tuberosity from a tilted 3-mm hole
preliminary drilled through the cortical layer into the bone
canal and then was pushed down to the distal metaphysis.
The opposite wire tip at the entrance point was cut, bent
like a loop, placed under the segment fascia, and the soft
tissues were sutured over it. Introduction of the wire did not
required additional reaming (Fig. 1). The wires remained
in the bone marrow canal during the entire experiment and
were removed after four weeks.
Upon completion of euthanasia, 1-cm-high diaphyseal
fragments were sawn out. They were fixed, decalcified and
dehydrated according to a standard method and then
embedded into celloidin. Transverse histotopographic
sections, 20?24 lm thick, were obtained with a sledge
microtome (Reichard, Germany), stained with hematoxylin
and eosin and according to Van Gieson. Histological bone
tissue preparations were studied with a large microscope
(Carl Zeiss Opton, Germany). A software complex
DiaMorph (DiaMorph, Russia) was used for morphometric
study of the bone tissue content in the bone marrow canal
and the thickness of the envelope formed around the
intramedullary wire. Volume of bone tissue (%) in the
digital images was defined with an analyzing software
program VideoTest-Morfologia (St. Petersburg, Russia).
Concentrations of Ca and P in the transverse
microsections of the diaphysis, embedded in araldite, were assessed
using an X-ray electron probe analyzer INKA Energy 200
(Oxford Instruments Analytical, UK) that is adjusted to a
Fig. 1 Fixation of an intramedullary wire with a formed end loop
scanning electron microscope JSM-840 under an
accelerating voltage of 20 kV and operation state of 15 mm. The
results were element maps and findings of their quantities
in weight percentage.
Osteointegration was also assessed quantitatively by
measuring the pull-off force with which the intramedullary
wire was removed out of the medullary canal. The
measurements were performed with a tensile sensor
dynamometer DEPZ-1D-1U-1 (MeDeTal, Russia) that
provides a measurement accuracy of ?0.01 N.
Statistical processing of the quantitative findings
obtained was performed using a paired double-sample t test
(p \ 0.05) and Wilcoxon test for independent samples
(p \ 0.05).
The study was approved by the ethics board of the
institution. Interventions, animal care and euthanasia
conformed to the requirements of the European Convention for
the Protection of Vertebrate Animals used for
Experimental and other Scientific Purposes (Strasbourg, 18.03.1986);
Principles of Laboratory Animal Care (NIH publication
No. 85-23, revised 1985), as well as the national laws.
Clinical examination of the animals during the experiment
did not reveal any changes in their general state, food or
water consumption. No neurologic or infection
complications were observed. Weight bearing was complete.
Radiographic studies did not show any wire displacement
in all the dogs.
Microscopic study of wire surfaces
The surface of a stainless steel wire (SSW) featured
transverse scratches, 7 9 2 lm long, 2 9 5 lm wide and
4 9 7 lm deep. The distance between the notches was
from seven to 35 lm. The surface between the scratches
was smooth (Fig. 2a).
The surface of a titanium wire (TW) featured a less
expressed relief but a greater roughness that was uniform
along its surface (Fig. 2b). The diameter of concaves and
protuberances was from 0.4 to 1 lm.
The surface of HA-coated wires had round pours that
were positioned on the entire surface of the coating and HA
??waves.?? The shape and location of such ??waves?? differed
between the HA-coated SSW and HA-coated TW (Fig. 2c,
d). Concentric spindles were formed round the pours on the
surface of SSWs that united into a conglomeration, while
the HA spindles on the TW surface were not linked to the
pours but formed a trabecular-like picture where the crests
of more protruded parts were interchanging with the pours
and less protruded parts of HA coating. Globular structures
that had a diameter from one to 4 lm were diffusely
positioned on the coating of TWs. The pours of HA-coated
wires also differed. HA-coated SSWs had pours that were
from one to 4 lm in diameter with the distance of two to
7 lm between them. The pour openings on the TW coating
were more variable with the diameter from one to 25 lm.
The distance between them was not uniform and measured
from two to 25 lm.
The content (weight percentage) of Ca, P and Ca/P ratio
in the coating differed inconsiderably between the
HAcoated wire types (Table 1). Concentrations of Ca and P
were by 2.15 and by 5.7 lower in the coating of the TWs,
respectively (p \ 0.05). Ca/P ratio did not differ
significantly (p C 0.05).
We revealed significant differences in the diaphyseal
bone structure between the groups after 4 weeks (Fig. 3).
Widened Haversian canals were observed in the cortex of
all dogs, but they were more expressed in groups 3 and 4.
Trabecular bone in the bone marrow canal enveloped the
wires like a muff and was seen in all the cases. An
additional endosteal bone formation was also noted but was
more expressed in groups 3 and 4.
Osteogenic activity was lower in group 1. Its envelope
was discontinuous and was located over a connective tissue
capsule. Its thickness ranged from 0.25 to 0.3 mm.
Endosteal osteogenesis was week. The connective tissue capsule
was absent in group 2, and the envelope thickness was
between 0.4 and 0.6 mm. The endosteal response was more
expressed than in group 1.
The connective tissue capsule over the wires was absent
in groups 3 and 4. HA-coated wires induced a greater
endosteal osteogenesis as far as more volume of bone
tissue was present in the bone marrow canal. The muff
thickness was between 0.8 and 0.9 mm in group 3, while in
group 4 it measured between 1.2 and 1.5 mm. Maximum
percentage volume of trabecular bone was present in the
bone marrow canals of group 4 dogs that corresponded to a
mean of 27.1 ? 0.14% that was 16.9% higher than in
group 2 (p \ 0.05) (Fig. 4). It measured 17.3 ? 0.063%
from the total volume of the bone marrow canal in group 3
dogs that was by 9.8% lower than in group 4 (p \ 0.05)
and by 10.6% lower than in control group 1 (p \ 0.05).
The normal mean weight percentage of Ca in the canine
cortex is 22%. The findings in Table 2 show that the
outflow of Ca from the compact layer of groups 1 and 2 did
not differ considerably as compared with groups 3 and 4
(2.5 and 2.28%, respectively). There was no considerable
difference in the values between groups 3 and 4.
The content of osteotropic elements in the bony muff
that was formed around the wires was the least in group 1
(Table 2). Weight percentage of Ca and P in the bone muff
around TWs was higher in group 4. However, the Ca/P
ratio did not have any statistical difference between TW
Fig. 2 Surface microrelief:
a SSW; b TW; c HA-coated
SSW; d HA-coated TW. SEM,
mag. a 9300; b 9600;
c 91600; d 9900
groups. When groups 1 and 3 were compared, the content
of Ca and P in the bone muff was increased 1.6-fold and
1.4-fold, respectively, in group 3. The increase in Ca and P
was 1.54-fold and 3.44-fold higher in group 4 as compared
with group 2, respectively. Moreover, the Ca/P ratio was
close to a canine mature bone value in group 4.
Mechanical tests for measurements of the wire pull-off
strength provided the quantitative data on the degree of
wire osteointegration (Table 3). As it is seen in the table,
the least pull-off force was observed by removal of the
uncoated SSW. The microporous HA-coated TW surface
increased the bonding force as compared with the uncoated
TW. The force to pull out the HA-coated TW was 40%
greater as compared with SSW in group 1.
All the metals and their alloys that have been used as
implants or temporary fixators can be assessed in regard to
their impacts on the living tissues. There are biotolerant
materials such as stainless steel and cobalt chrome alloys,
or bioinert materials such as titanium oxides and
aluminum. Bioactive metals that would accelerate
osteogenesis have not been known yet.
The main role of current plating and nailing methods is
the mechanical retain of bone fragments in a reduced
position until bone unites . The loading is shunted to
the plate or nail when a person ambulates. It is considered
to be one of the drawbacks of these technologies as far as
the main complications that develop are osteosynthesis
instability and osteoporosis. Therefore, the solutions that
would be able to enhance stability and bone reparation
process by using nailing or plating have been under
investigation [6, 10].
Ca?P ceramics (CaPC) and bioglass have been studied
for possible use in bone tissue engineering. They have been
characterized by tight chemical links with the bone (bone
bonding) as well as by induction of bone formation on the
implant surface [11, 12]. However, the CaPC that features
an undoubted biological activity is too brittle to be used as
a massive material of a sufficient volume that could endure
much loading. Therefore, it cannot be applied as an
independent implant . It was found possible to coat the
surfaces of metallic implants with CaPC or HA with the
objective to combine the mechanical strength of the metal
with the Ca?P bioactivity [11, 13?15]. The MAO technique
enables to produce a qualitative and sufficiently thick
nanocomposite HA coating on implants as compared with
the RF sputtering technique that also reduces HA particles
to nanoscale but a HA layer is of a lesser thickness [13?15].
The HA coating produced with the MAO technique on
titanium surfaces has attractive properties, such as high
porosity, a controllable thickness and a considerable
Fig. 3 Osteointegration 1 month after intramedullary wires
introduction: a, e group 1; b, f group 2; c, g group 3; d, h group 4. Upper
row histotopographic sections. Hematoxylin and eosin staining. Mag.
Fig. 4 Percentage of bone tissue in the bone marrow of all groups
canal of the tibia after 4 weeks of the experiment
density, which favor its use in dental and bone surgery
[14, 15]. Moreover, the MAO technique is easier and
cheaper for fabrication.
We have hypothesized that intramedullary
osteosynthesis could be realized with HA-coated wires available on
our market and started to use them instead of regular nails
in clinical practice for reinforcement of bone fragments
stability in diaphyseal fracture repair, bone lengthening and
deformity correction in combination with the Ilizarov
external fixation [6?9]. We observed that bone formation
91.5. Lower row maps of X-ray electron probe microanalyzer by
characteristic calcium radiation. Mag. 920
and repair went on faster with their use . Therefore, we
designed the experiment to study the effect of wires for
diaphyseal fracture repair and found that facture healing
was 1.6 times faster in the series with the combination of
the techniques [6, 9]. The current study, being the
continuation of the previous experiments of the possible ways to
fasten osteointegration of wires, reveals not only the
phenomena that undergo by introduction of the HA-coated
wires into the medullary canal versus uncoated ones but
also the degree of osteointegration by measuring the
pulloff strength .
Microscopic, histological and biomechanical tests
conducted during this experiment were aimed at an objective
quantitative estimation of the HA-coated wires bioactivity
in regard to osteoinduction and osteoconduction that may
result in osteointegration. They revealed that the SSWs
induced the least osteogenic activity in the bone marrow
canal. It was found that HA coating stimulates bone
formation around both SSWs and TWs. The implants were
enveloped into an osseous muff and provided integration
with bone tissue.
As the biomechanical tests showed , such wires got
tightly fixed in the bone tissue of the muff formed around
them. Other studies also assessed the bonding strength of
various biomaterials with the bone and found that treatment
methods are essential for preparing bioactive titanium
[5, 17, 18]. In our opinion, the pull-off of the wire
integrated into the bone tissue block is also influenced by the
bonding of the coating with the wire surface. It may be
conditioned by the features of the wire?s surface. We have
Table 2 Content of osteotropic elements in the tibia four weeks after the experiment (wt%)
* p \ 0.05 as compared with the wire type without HA coating
? p \ 0.05 as compared with the other HA-coated wire group
Italic values p \ 0.05 as compared between the groups without HA coating
Bold type p C 0.05
Table 3 Pull-off strength of
* p \ 0.05 as compared with a corresponding wire type without coating
revealed that the roughness that was more expressed in
case of TWs as compared with the SSWs added to the
bonding with HA coating. Finally, it contributed to a
special ??relief?? of the surface due to coating that enhanced
osteointegration properties of the titanium implant and
formed a stronger implant-to-bone tissue block.
It is well known that one of the main drawbacks of the
standard intramedullary osteosynthesis is the risk of
damaging the medullary canal content, mainly its vessels .
It results in a weaker ability of pluripotent bone marrow
stromal cells to osteogenesis and osteoinduction. As a
matter of fact, the introduction of intramedullary wires
almost excludes the damage to the intraosseous artery or
injury to the endosteum as far as the wires we use have a
bunt end, are of a small diameter (1.8?2.0 mm) and occupy
not more than 30% of the canine bone marrow canal. In
humans, they take a far more less space as even the
narrowest part of the canal is wider. We did not have any
arterial damage in our clinical practice [7, 8].
Moreover, a prolonged formation of local granulation
foci in the bone marrow cavity could also have a
stimulation effect. Local granulation foci that are created by
endocrinic and paracrinic ways provide the increase in
osteoproductive cells in the fracture area, stimulate
regenerative angiogenesis and thus promote activation of
osteoreparation. In such conditions, fracture repair runs
faster and is of primary type without cartilaginous or
connective tissue by bone bonding [3, 20]. The Ca/P ratio
in the HA coating was less than 1.0. We believe that it also
played a definite role in the launch of osteoconduction
process as it was similar to the one that is observed by
sedimentation of bone morphogenetic proteins on the
collagen matrix, or in other words, to the initial stage of new
bone tissue mineralization.
Being low invasive, the intramedullary osteosynthesis
with HA-coated wires fulfills two main functions. First, it
is able to provide an additional stability of bone fragments
due to a fast osteointegration at the expense of the bony
envelope formed. And second, it creates osteoconductive
and osteoinductive conditions for bone tissue reparative
regeneration due to a HA-based biomaterial. The method of
intramedullary reinforcement with bioactive HA-coated
wires stimulates the activity of bone marrow and provokes
endosteal bone formation.
The performance of a HA-coated implant depends on its
coating properties (thickness, porosity, HA content,
crystallinity) and implant roughness . It appeared that the
HA-coated surface relief architecture and pours of our
experimental wires did not provoke formation of
connective tissue. Instead, the bone marrow canal was filled in
with spongy bone which was sufficiently dense round the
implant and provided its osteointegration.
We believe that the effect of such wires could be
successively used for management of patients with diaphyseal
fractures, osteogenesis imperfecta, phosphate diabetes, as
well as in cases when large amounts of lengthening are
required . Further research and clinical tests in other
orthopedic situations (bone defects and lengthening) are
Acknowledgements The study was conducted under financial
support of the Russian Science Foundation (Project # 16-15-00176).
Compliance with ethical standards
Ethical approval All applicable international, national, and/or
institutional guidelines for the care and use ofanimals were followed.
Informed consent Informed consent is not applicable in this study.
Open Access This article is distributed under the terms of the
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