ORIGINAL ARTICLE
Xenograft Bone Inclusion Improves Incorporation of
Hydroxyapatite Cement Into Cancellous Defects
Michael J. Voor, PhD, Eric M. Yoder, BS, and Robert L. Burden, Jr, MEng
Objectives: Hydroxyapatite cement (HAC) is biocompatible and
osteoconductive, but its slow resorption limits new bone formation.
The purpose of this investigation was to determine the effects of
adding partially demineralized xenograft cortical bone in an
established animal model.
Methods: Eight 6-month-old female New Zealand white rabbits
were used. Drill-hole defects (8.0 mm long, 5.0 mm diameter) were
prepared and filled with either HAC alone or HAC mixed with
processed xenograft bone particles from young pigs (XBC) at
a volumetric ratio of approximately 25%. The particles were
approximately 5 mm long and 1 mm diameter and were extensively
washed, demineralized in dilute hydrochloric acid, and rewashed.
Microcomputed tomography scanning and histology were performed
after 10 weeks. New bone and inflammatory/immune response were
graded on a 0 to 3 scale and calcein labeling was quantified as percent
area new bone. Statistical analyses were by Student’s t tests.
Results: XBC showed significantly more new bone formation than
HAC throughout the defect (P , 0.05). XBC also showed
significantly more inflammatory/immune response than HAC (P ,
0.05). The three-dimensional microcomputed tomography recon-
structions showed that the HAC was basically inert, whereas the XBC
took on an appearance suggestive of more extensive incorporation.
Conclusions: Adding xenograft to HAC creates a bioactive
composite that is more rapidly incorporated, resorbed, and replaced
by new bone. The presence of processed xenograft particles creates
a vigorous inflammatory response, but there may be some benefit to
the resorption rate of the HAC as a result of the infiltration of cells.
Future research should focus on the longer-term incorporation and
remodeling of XBC.
Accepted for publication May 16, 2011.
From the University of Louisville Orthopaedic Bioengineering Laboratory,
Louisville, KY.
M.J.V. has received research funding from the Kentucky Science and
Engineering Foundation (Grant # KSEF-148-502-05-357) to support this
work. M.J.V. has ownership interest in a private company, Vivorte Inc, that
has licensed technology related to this work from the University of
Louisville. There are no disclosures for Eric Yoder and Robert Burden.
This study was presented in part at the Annual Meeting of the Orthopaedic
Trauma Association, Baltimore, MD, 2010.
Reprints: Michael J. Voor, PhD, Associate Professor, Department of
Orthopaedic Surgery, University of Louisville, Louisville, KY 40292
(e-mail: mike.voor@louisville.edu).
Copyright Ó 2011 by Lippincott Williams & Wilkins
J Orthop Trauma  Volume 25, Number 8, August 2011
Key Words: bone graft, calcium phosphate, hydroxyapatite, bone
substitute, xenograft
(J Orthop Trauma 2011;25:483–487)
INTRODUCTION
Hydroxyapatite cement (HAC) and other calcium
phosphate cements (CPCs) are biocompatible and osteocon-
ductive, but their slow resorption limits new bone formation.1,2
The addition of pores or biologic materials helps resorption
and incorporation but typically compromises short-term
strength.3 The purpose of this investigation was to determine
the effects of adding partially demineralized xenograft cortical
bone on cement resorption and new bone formation over
10 weeks in an established animal defect model.
Recent advances in processing of allograft bone have
decreased the immune and inflammatory response as a result
of the removal of all of the cellular and most of the other
biologic materials except the matrix proteins and mineral.4–6
Several new products such as Pro-Stim (Wright Medical,
Arlington, TN) and EquivaBone (Etex Corporation, Cam-
bridge, MA) now include fully demineralized allograft bone
with a synthetic ceramic cement to form a composite bone
substitute material with advantages over purely synthetic or
purely biologic materials. If similar treatments are applied to
xenograft bone, one should expect a similar decrease in the
immune and inflammatory response relative to that which has
been observed with fresh or merely frozen xenografts.
Examples already exist, in the form of Type I collagen
scaffolds, for the success of highly processed xenograft for
bone graft applications. Other potential clinical applications
for xenograft bone exist in the bone graft substitute field,
where the potential advantages over allograft are larger supply,
greater source control, and lower costs. If partially deminer-
alized xenograft bone can be included in a ceramic bone
substitute material with a sufficiently low level of negative host
response, it should be capable of functioning as an acceptable
bone graft as well. The hypothesis was that adding processed
xenograft bone particles would increase incorporation and
formation of new bone within the new composite cement
material.
MATERIALS AND METHODS
Cement Preparation
Two different CPCs were used for this experiment.
The first was commercially available HAC that was used as
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Voor et al
a control (BoneSource; Stryker, Mahwah, NJ). The second
was an experimental bone cement (XBC) consisting of
BoneSource mixed with specially processed porcine
cortical bone particles. These xenograft particles were
prepared as elongated ‘‘needles’’ of cortical bone approx-
imately 5 mm long and 1 mm diameter that were extensively
washed using an organic detergent (Triton X-100),
demineralized in dilute hydrochloric acid, rinsed, and
rewashed. The particles were allowed to dry over night in
an oven at 37°C and then added to the cement powder at
a volumetric ratio of approximately 25%. This ratio was
chosen as a balance between the desire to accelerate the
biologic activity of the composite material (more bone) and
to maintain the handling and strength characteristics of the
cement (less bone). A volume fraction of 25% is also in the
midrange for the cancellous bone being treated in this
model and thus should allow for restoration of the
trabecular architecture. For consistency and efficiency in
surgery, premolded cylinders (5 mm diameter, 8 mm length)
of both HAC and XBC were prepared in batch form and
allowed to harden in sterile containers at 37°C and
approximately 100% humidity.
Surgery
All procedures were approved by the Institutional Animal
Care and Use Committee (IACUC). Eight 6-month-old female
New Zealand white rabbits weighing approximately 4 kg were
FIGURE 1. Rabbit femurs were
scanned at 14-mm voxel resolution.
After 10 weeks, the HAC was well
attached to host bone but was
a homogeneous solid. The XBC
was heterogeneous with apparent
resorption and new bone. HAC,
hydroxyapatite cement; XBC, exper-
imental bone cement.
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J Orthop Trauma  Volume 25, Number 8, August 2011
TABLE 1. Inflammatory and Immune Response Grading Scale
Score Description
0 No sign of cellular activity
1 Cellular activity only at defect boundary
2 Cellular activity at defect boundary with areas of interior cellular
activity and fibrous tissue
3 Cellular activity at defect boundary with greater than 20% of the
interior area demonstrating cellular activity and fibrous tissue
TABLE 2. New Bone Formation and Material Incorporation
Grading Scale
Score Description
0 No activity except peripheral attachment
1 No central activity, cement resorption and new bone infiltration
only at boundary
2 Infiltration of new bone at defect boundary and some interior areas
of new bone formation and cement resorption activity
3 Infiltration of new bone at defect boundary and greater than 20% of
interior area showing new bone formation and cement resorption
activity
used. Bilateral drill-hole defects (8.0 mm long, 5.0 mm
diameter) were prepared in the distal femoral lateral condyles
based on an established protocol.7,8 At random, the right and left
defects were filled with either HAC or XBC. After placement of
Ó 2011 by Lippincott Williams & Wilkins
J Orthop Trauma  Volume 25, Number 8, August 2011
FIGURE 2. After 10 weeks, there was no new bone
formation within the HAC-filled defect (above left),
whereas the XBC-filled defect had several regions of
extensive cellular activity with new bone formation.
HAC, hydroxyapatite cement; XBC, experimental
bone cement.
the cement cylinder in the defect, the opening of the defect was
covered with bone wax. The incisions were closed in layers and
the rabbits were allowed to recover and ambulate as tolerated.
They had access to food and water ad libitum.
Evaluation
Three days before euthanasia (at 10 weeks postopera-
tively), each rabbit was given a subcutaneous injection of calcein
solution to label for new bone formation. After euthanasia, each
femur was prepared for microcomputed tomography scanning
Ó 2011 by Lippincott Williams & Wilkins
Xenograft Bone Inclusion Improves Incorporation
and decalcified and undecalcified histology. Decalcified speci-
mens were stained with hematoxylin and eosin and the
inflammatory/immune response as well as new bone formation
and defect incorporation were graded on a 0 to 3 scale (Tables 1
and 2). The undecalcified specimens were prepared for
fluorescence microscopy and calcein labeling was quantified
as percent area of new bone.
Because there were only two groups for comparison,
statistical analyses of all measurements were by Student’s t tests.
Differences were considered statistically significant at P , 0.05.
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Voor et al
FIGURE 3. There was significantly more inflammatory/cellular
response in the XBC compared with HAC (P , 0.05). HAC,
hydroxyapatite cement; XBC, experimental bone cement.
RESULTS
The three-dimensional microcomputed tomography
reconstructions showed qualitatively that the HAC was
basically inert, whereas the XBC took on an appearance
suggestive of more extensive incorporation (Fig. 1). The
incorporation of the XBC was confirmed by the decalcified
histology (Fig. 2), which showed that there was little to no
activity in the HAC, whereas there were large regions of the
XBC that were resorbed and replaced by new bone with
extensive cellular activity. Quantitatively, the XBC showed
significantly more inflammatory/immune response than HAC
(P , 0.05) (Fig. 3) and XBC showed significantly more new
bone formation than HAC throughout the defect (P , 0.05)
(Fig. 4). Calcein labeling of the undecalcified samples also
showed larger regions of active new bone formation in the
XBC-filled defects compared with the HAC-filled defects (P =
0.06) (Fig. 5). These quantities did not reach statistical
significance but only represented the last 3 days of new bone
formation, not the total amount.
FIGURE 4. After 10 weeks, there was significantly more new
bone formation within the XBC defects compared with HAC
(P , 0.05). HAC, hydroxyapatite cement; XBC, experimental
bone cement.
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J Orthop Trauma  Volume 25, Number 8, August 2011
DISCUSSION
The advantage of the injectable CPCs as bone void
fillers is that they can flow and mold to fill defects of any shape
and then harden to provide immediate mechanical stability
with excellent biocompatibility. The disadvantage of these
materials has been that they are very slow to resorb and
incorporate. Clinical success of CPC as a bone void filler has
therefore been tempered by concerns that the material will not
be readily replaced by new bone.
Several attempts have been made to increase the
incorporation rate of CPCs by substituting calcium phosphate
material with something more easily resorbed or by simply
increasing porosity. However, for such approaches to be
successful, the new material should at least be as strong as the
original CPC in the short term when healing of the defect is
taking place. This study investigated a simple and inexpensive
approach involving xenograft cortical bone particles because
they are as strong as CPC and have some advantages with
respect to resorption.
It is well known that the CPCs are similar chemically to
bone mineral. As such, the CPC material is often observed to
have new bone formation directly on its surface. Incorporation
only takes place through a direct cellular resorption
(osteoclastic) response.1,9 The slow remodeling process often
results in a continuous mass of CPC fully coated in new bone,
but not rapidly replaced by new bone.
Previous attempts to accelerate the incorporation pro-
cess have led to the conclusion that the bulk of the material
must be infiltrated with pathways or channels for the rapid and
complete infiltration of the cement with active cells.3,8–11 The
XBC material described in this investigation accomplishes this
goal with elongated particles of partially demineralized
xenograft cortical bone. Importantly, in its initial condition,
XBC has similar mechanical strength to the CPC on which it is
based. However, the partially demineralized bone particles not
only resorb rapidly when exposed to the cellular infiltration
from the host, but they elicit a slightly increased inflammatory
response, which likely accelerates the cell-mediated resorption
of the CPC itself. This effect is obvious in Figure 4 in the large
region of cellular fibrous tissue, new bone formation, and
a wavefront of resorption of the CPC.
The limitations of this investigation are that it only
represents a single time point postimplantation of the XBC. It
is not known whether the HAC component would be fully
resorbed over time or whether there would be an unacceptable
inflammatory response that may negate any possible resorptive
benefit. Future studies of xenograft as an alternative to improve
the rate of resorption and incorporation of CPC should include
complete immunologic assays to determine if there should be
any concerns about the host response to the xenograft chosen
for a particular application. Also, multiple time points and
longer time points should be tested to fully develop the time
course of the strength of the bone void filler and to determine
the completeness with which the CPC would be expected to
resorb. Additional studies could also determine the maximum
and minimum ratios of bone to cement which allow for
acceptable handling characteristics and sufficient resorption,
respectively.
Ó 2011 by Lippincott Williams & Wilkins
J Orthop Trauma  Volume 25, Number 8, August 2011
FIGURE 5. After 10 weeks, there was
a trend (nonsignificant [NS]) toward
more new bone formation activity
within the XBC defects as indicated
by calcein labeling compared with
HAC. Note that the highlighted
regions only show new bone forma-
tion in the last 3 days. HAC, hydroxy-
apatite cement; XBC, experimental
bone cement.
Adding xenograft to HAC creates a bioactive composite
that is more rapidly incorporated, resorbed, and replaced by
new bone than pure HAC. The presence of xenograft particles
creates a marked inflammatory response, but there may be
some benefit to the resorption rate of the HAC component of
the XBC as a result of the rapid infiltration of cells. Future
research should focus on the longer-term incorporation and
remodeling of XBC to determine if complete resorption is
facilitated.
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Xenograft Bone Inclusion Improves Incorporation
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