Journal of Biomaterials and Nanobiotechnology, 2011, 2, 15 0-155
doi:10.4236/jbnb.2011.22019 Published Online April 2011 (
Copyright © 2011 SciRes. JBNB
In Vivo Evaluation of Functionalized Biomimetic
Hydroxyapatite for Local Delivery of Active
Johan Forsgren1, Ulrika Brohede2, Sonya Piskounova3, Albert Mihranyan1, Sune Larsson4,
Maria Strømme1, Håkan Engqvist1
1Department of Engineering Sciences, Uppsala University, Uppsala, Sweden; 2Sandvik AB, Stockholm, Sweden; 3Department of
Materials Chemistry, Uppsala University, Uppsala, Sweden; 4Department of Orthopaedics, Uppsala University Hospital, Uppsala,
Received November 3rd, 2010; revised January 19th, 2011; accepted January 22nd, 2011.
This study was carried out to investiga te the biological respo nse in vivo to biomimetic hydroxyapatite implant coatings
functionalized with bisphosphonates and bone morphogenetic proteins. The functionalization was carried out by a sim-
ple soaking procedure in the operating room immediately prior to surgery. Cylindrical titanium samples with and
without coatings were implanted in the distal femoral epiphysis of sheep and retrieved after 6 weeks. The histological
analysis proved tha t all samples were integrated well in the tissu e with no signs of intolerance. Fewer osteoclasts were
observed in the vicinity of bisphosphonate-functionalized samples and the bone was denser around these samples com-
pared to the other samples. Samples functionalized with bone morphogenetic protein induced more bone/implant con-
tact but showed a more inconsisten t outcome with reduced bone density around the samples. This study demonstrates a
simple method to functionalize implant coatings, which provides surgeons with an option of patient-specific function-
alization of implants. The ob served biologica l impact due to the delivery o f active molecules from the coatings su ggests
that this strategy may also be employed to deliver antibiotics from similar coa tings.
Keywords: Implant, Titanium, Hydroxyapatite, Bisphosphonate, Bone Morp hogenetic Protein-2
The success rate of hip and knee arthroplasties is high;
more than 90% of the patients are predicted to live more
than 10 years with their implant before revision surgery
is needed [1,2]. But despite the high success rate, the
number of revision surgeries is still far from satisfactory.
Only in the U.S., 36,000 revision total hip arthroplasties
and 32,700 revision total knee arthroplasties were per-
formed in 2003, and the annual number is expected to
increase steadily [3]. Revision surgeries due to implant
failure are often complicated, expensive and painful for
afflicted patients. In a stud y from 1995 it was shown that
patients undergoing revision orthopedic surgery need to
stay longer at the hospital and the time in the operating
room is significantly higher compared with primary sur-
gery [4]. In two retrospect studies of revision hip and
knee arthroplasties, as much as 39% of the hip surgeries
[1] and 63% of the knee surgeries [5] were performed
within 5 years following primary surgery. Infection and
aseptic loosening are two common indications for early
implant failure of uncemented prostheses [1,2]. Surgical
site infections account for 38% of all nosocomial infec-
tions in the US [6] and the incidence rate can be as high
as 10% at spinal surgery [7]. In a recent retrospect study
of 9245 patients undergoing primary hip or knee arthro-
plasty, 65% of the developed infections appeared within
one year after primary surgery [8]. These findings show
that perioperative infections associated with the surgical
wound is a challenge that needs to be addressed if the
number of revision surgeries is to be reduced.
The interface between prosthetic implant and tissue is
known to be a region of local immune depression with
reduced resistance to microbes, often referred to as an
immune-incompetent fibro-inflammatory zone [9]. In
addition, implant migration due instability in the inter-
face between bone and implant can damage the tissues
In Vivo Evaluation of Functionalized Biomimetic Hydroxyapatite for Local Delivery of Active Agents 151
and almost completely deplete the immune defenses [10].
Thus, these peri-prosthetic regions are highly sensitive
for bacteria colonization and biofilm formation. To reduc e
the risk for bacteria colonization with consecutive de-
velopment of infection, it is desirable with bactericidal
effect on the implant surface and a firm fixation of the
prosthesis. This could be obtained by local and sustained
release of active agents from the implant surface that
promotes more dense bone formation around the pros-
thesis and/or has a bactericidal effect. It has been shown
that the number of early failures associated with poor
implant fixation have been reduced when osteoconduc-
tive coatings have been deposited on the implant surfaces
[11,12]. Hydroxyapatite, a calcium phosphate material
resembling the mineral phase found in bone [13], is the
most studied and applied osteoconductive material, and
earlier in vitro studies have proven the feasibility of
biomimetic hydroxyapatite (BHA) as carrier of various
active agents such as antibiotics [14], bisphosphonates
(BIS) [15] and bone morphogenetic proteins (BMP) [16]
for targeted administration at the site of implant. In the
present study, the possibility of incorporating various
agents to a BHA coating by a rapid on-site loading
method was evaluated in vivo. The aim is to provide su r-
geons with an option to choose on-site if and what type
of drug should be incorporated into the coating. In the
design of the study, BIS and BMP were chosen, as they
affect the bone formation surrounding the implant, which
easily can be analyzed. The effect of antibiotics would be
on the inhibition of bacteria entering the incision wound
and such study would be more complicated to perform
and more difficult to defend from an ethical standpoint as
it would involve a subs tantial risk for the animals.
2. Materials and Methods
2.1. Implant Preparations
Cylindrical implants of titanium grade 2 with 8.0 mm
length and 3.5 mm diameter were used in this compara-
tive study, see Figure 1. The implants were delivered
with a mounting device used to screw th e implants in the
right position, this part was subsequently removed after
fixation of the implants. Three different surface modifi-
cations of the implants were evaluated and compared to a
native titanium surface, see Table 1. Out of the total 20
implants that were prepared, 5 were left non-coated and
15 were coated with a layer of BHA. Preparation of the
BHA coatings was performed as descried in earlier work
[17]. Briefly, a layer of anatase TiO2 was deposited on
the titanium cylinders using physical vapor deposition
(PVD) before the samples were immersed in 40ml Dul-
becco’s phosphate buffered saline (Dulbecco’s PBS,
Sigma-Aldrich) each for 4 days at 60. The samples
were subsequently retrieved and analyzed with scanning
Figure 1. Photograph of implant where the cylindrical pin
constitutes the actual sample, the screw at the top was used
to fixate the implant in the cortical bone.
Table 1. Names and corresponding surface characteristics
of the different sample.
Sample typeNo. of samplesSurface
Control 5 Native titanium
Test 1 5 BHA coating
Test 2 5 BHA coating loaded with BMP-2
Test 3 5 BHA coating loaded with BIS
electron microscopy (SEM) to examine the precipitated
layer of BHA. It was observed that the BHA-layer was
not fully covering the cylinders and therefore the im-
plants were once more immersed in PBS for additional 7
days at 37. BHA-coatings prepared in a similar way
have been described earlier [17-19] and have been shown
to be highly porous and calcium deficient compared with
stoichiometric HA, something that promotes incorpora-
tion of bioactive molecules [14,19]. After deposition of
the BHA-layer, the implants were rinsed in deionized
water, dried in air and sterilized by autoclaving (30 min,
120). In the operating room, 5 BHA-coated implants
were placed in individual sterile plastic tubes containing
0.5 ml of an autoclaved aqueous solution of the bisphos-
phonatePamindronate (0.5 mg/ml, Sigma-Aldrich). An-
other 5 BHA-coated implants were soaked in a 0.5 ml
recombinant human BMP-2 solution (rhBMP-2, 0.15
mg/ ml , I n d u ct O S ®, Wyeth Europe) in formulation buffer
containing 2.5% glycine, 0.5% sucrose, 0.01% Polysor-
bate 80, 5 mM sodium chloride and 5 mM L-glutamic
acid. The implants were immersed in the solutions for 10
- 30 minutes before implantation.
2.2. Animal Study
The in vivo study was designed for intra-osseous implan-
tation in sheep for 6 weeks. Five 24-month-old female
Grivette sheep, weighing from 51 to 63 kg, were used in
the study. Prior to the surgical procedure, the animals
were fasted overnight. At the time of implantation,
pre-medication and anesthesia was performed by intra-
venous injection of a thiopental-pentobarbital mixture
(Nesdonal®, Merial; and Pentobarbital Sodique, CEVA
Santé animale) and atropine (AtropinumSulfuricum,
opyright © 2011 SciRes. JBNB
152 In Vivo Evaluation of Functionalized Biomimetic Hydroxyapatite for Local Delivery of Active Agents
Aguettant) followed by the inhalation of an O2-isoflurane
(1% - 4%) mixture (Afrane®, Baxter). Each animal re-
ceived a first analgesic (Flunixine, Meflosyl®, Fort
Dodge Santé Animale) and as a prophylactic measure,
perioperative antibiotics penicillin procaine and penicil-
lin benzathine (Duplocilline®, Intervet) were given. In
addition, each animal received a second analgesic (Bu-
prenorphine, Temgesic®, Schering Plough) preoperat ively.
Bone defects were created bilaterally in the distal
femoral epiphysis and the sites were implanted with
samples. Each animal was implanted with 4 samples, one
of each type. An orthopedic drill was used to create cy-
lindrical defects with approximately 2.3 mm in diameter
by < 9 mm deep in the distal femoral epiphysis. Drilling
was performed under constant irrigation with saline solu-
tion (NaCl 0.9%) and suction. Intra-osseous implantation
was performed as recommended by the ISO 10993-6
standard (2007) with the cylindrical parts of the samples
inserted in the cancellous bone. Once implanted, the
samples were in contact with bone along the sides while
the bottom was facing a cavity in the bone. This enabled
a study of how bone is formed around the implan ts under
different conditions. The animals were treated and kept
according to the conditions conformed by the EU re-
quirements of farm animals (EC Directive 86/609). Dur-
ing the observation period, penicillin procaine and peni-
cillin benzathine (Duplocilline®, Intervet) were continued
on days 3, 6 and 9 following surgery. The analgesic
(flunixine, Meflosyl®, Fort Dodge Santé Animale) was
administered every other day for one week following
surgery. After a 6-week implantation period, the animals
were sacrificed by lethal injection of a barbiturate
(DolethalND, Vetoquinol) and implanted sites were sam-
The protocol for the study was approved by
BIOMATECH (France) Ethical Committee on October
14, 2008.
2.3. Sample Preparation
The retrieved samples were dehydrated in ethanol solu-
tions of increasing concentration, cleared in xylene, and
embedded in poly-methyl methacrylate (PMMA). One
longitudinal section in the long axis of the implant was
obtained per specimen where one half of the sample was
sectioned by a microcutting and grinding technique
adapted from Donath et al [20]. The remaining half from
each specimen was sputtered with a thin gold/palladium
layer to enable SEM analysis.
2.4. Histology
Sections were stained with modified Paragon for semi-
quantitative and qualitative analyses. Histological analy-
ses were performed using a Nikon microscope coupled
with a digital camera (magnifications of x4, x10, x20 and
x40). Semi-quantitative evaluation of local tolerance was
performed according to ISO 10993-6 standard.
2.5. Bone/Implant Contact and Adjacent Bone
The samples prepared for SEM analysis were analyzed in
backscatter mode using 15 keV acceleration voltage (Leo
1550 FEG Gemini, Zeiss). The bone/implant contact was
calculated from the images by measuring the contact
length along the periphery of the sides and bottom of the
implants, see example in Figure 2. Quantitative analysis
was performed on the SEM images using a software
(LeicaQwin) to calculate the bone density within the
nearest 200 m of tissue surrounding the implants. Areas
of soft tissue and bone along the sides and bottom of im-
plants were determined from the difference in contrast
between the two areas. The bone/soft tissue ratio was
calculated from these measurements.
3. Results and Discussion
No local signs of necrosis were observed macroscopi-
cally with either the test or control samples. Moderate
signs of hemorrhage were observed at the level of the
soft tissues surrounding the type 1 and type 2 samples.
No signs of exudates were observed with either the test
or control samples with exception of one animal, which
showed the presence of a subcutaneous liquid pocket in
the right and left femur graded as marked exudates and
increasing the mean grade of exudation similarly in all
No local intolerance signs were observed for any of
the samples except for one of the test 3 samples, where a
marked infiltration of lymphocytes without significant
implication on the bone healing was observed. Marked
grade of newly formed bone showing characteristics of a
mixture of lamellar and woven bone with osseous con-
densation were seen along the surface for all sample
types. In terms of healing performance, the histology
analysis demonstrated that the control samples and the
Figure 2. SEM-image of implanted sample (test 3). The im-
age shows bone around the e ntire sample.
opyright © 2011 SciRes. JBNB
In Vivo Evaluation of Functionalized Biomimetic Hydroxyapatite for Local Delivery of Active Agents 153
te s t 1 a n d t e s t 3 s a mp l e s y i e l d ed m a r k e d a n d s i mi l a r amount
of newly formed bone and level of implant osseointegra-
tion. Sites implanted with test 2 samples showed incon-
sistent outcome with a slightly lower healing perform-
ance as compared to the previous samples. Very few os-
teoclasts were observed in the vicinity of the test 3 sam-
ples compared to moderate numbers for the other types
of implants. Figure 3 shows active bone deposition on a
test 1 sample.
The results from the bone/implant contact measure-
ments are presented in Figure 4 as the average difference
in contact between the test samples and the control sam-
ple in respective sheep. The displayed results are based
on three values as the highest and lowest values were
excluded from the calculations. Notably is the large im-
pact of BMP-2 seen at the bottom of the samples. Here,
the implants were not in contact with bone at implanta-
tion and the BMP-2 clearly induces more bone formation
along the periphery of the implants at these conditions.
At the sides of the implant, the results for the test sam-
ples were equivalent to the control samples and no sig-
nificant difference could be seen. The apposition of new
bone around the samples was not improved by the BHA
coating as the control samples were equally integrated in
the bone a s the test 1 samp les with an aver age bone con-
tact of 62% on the sides and 52% at the bottom. The
BMP-2 had no detectable effect along the sides of the
implants. Here, the implants were in contact with bone
immediately after implantation and the different condi-
tions for healing and bone formation clearly affected the
impact of the delivered BMP-2. The test 2 samples were
the only ones with more bone apposition at the bottom
compared to the sides with about 20% more bone contact
at the bottom. Also notably is that the BIS did not affect
the bone contact.
Figure 5 displays the results from the bone density
measurements where the results are presented as the
Figure 3. Histologic image (×10) of active bone deposition
around an implant with BHA coating. The black area in the
lower part of the image is the implant, red color indicates
osseous tissues with osteocytes (OC), the blue linings sur-
rounding the bone are composed of active osteoblasts (OB)
and white areas indicate bone marrow (M). The sample was
stained with modified Paragon.
average difference in bone density between the test sam-
ples and the control sample in respective sheep. The re-
sults are based on three values as the highest and lowest
values were excluded from the calculations. Here, it can
be seen that the delivered BIS had a significant impact o n
the bone formation around the test 3 samples. This was
also confirmed by the histology analysis where very
slight numbers of osteoclasts were observed in the vicin-
ity of these implan ts.
The greatest impact was seen at the bottom, which
suggests that the delivered BIS was most effective in
areas in total absence of bone. Notably is the lower bone
density around the test 2 samples and this observation
may find its explanation in the dual effect of BMP-2 that
also activates apoptosis in postnatal osteoblasts [21,22].
BMP-2 has also been shown to decrease calcium deposi-
tion in vitro [22,23] and the findings in this study suggest
that the delivered BMP-2 actively promotes activation of
newly recruited mesenchymal stem cells and osteoblast
progenitor cells to produce bone directly at the HA co at-
ing while more mature osteoblasts already present in the
bone at some distance from the implant are stimulated in
a negative way. A higher concentration on BMP-2 in the
Figure 4. Average difference in bone/implant contact for the
test samples compared to the control sample in respective
sheep, error bars show the standard deviation for the three
Figure 5. Average difference in adjacent bone density for
the test samples compared to the control sample in respec-
tive sheep, error bars show the standard deviation for the
three samples.
opyright © 2011 SciRes. JBNB
154 In Vivo Evaluation of Functionalized Biomimetic Hydroxyapatite for Local Delivery of Active Agents
loading solution might have rendered a more effective
functionalization of the BHA coating, resulting in a
higher amount of BMP-2 delivered to the tissues. This
could have caused another biological response if the ef-
fect is dose-dependent. The BHA coating alone was not
observed to have any impact on the bone formation
around the implants after 6 weeks of implantation, which
is in line with earlier studies [24]. Yet, it is in con trary to
the positive effects of biomimetic HA on bone formation
observed by Barrère et al [25].
The findings in this study prove that it is possible to
obtain a positive effect on the bon e formation in vivo via
local delivery of active agents from implant coatings
comprised of BHA. In the future, this strategy of incor-
porating active molecules to BHA coatings in the oper-
ating room immediately prior to implant insertion is
suggested to be employed to deliver single drugs or com-
binations of drugs including, e.g., antibiotics from the
surfaces to reduce the incident rate of surgery-related
4. Conclusions
The study demonstrates an effective method to function-
alize implants immediately prior to surgery. The func-
tionalization procedure in the operating room did not
lead to any infections in the animals and the active
molecules delivered from the coatings were shown to
have an impact on the bone formation. BMP-2 was
shown to promote more bone formation directly along
the periphery of the implants in areas without bone and
BIS promoted more dense bone formation. The proposed
strategy is also suggested to be used for local release of,
e.g., antibiotics in the future to reduce the incident rate of
surgery-related infections.
5. Acknowledgements
The Swedish Research Council is acknowledged for
funding this study.
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