Infection Rates and Healing Using Bone Wax and a Soluble
Tadeusz Wellisz MD, Yuehuei H. An MD, Xuejun Wen MD, PhD,
Qian Kang MD, Christopher M. Hill VMD, Jonathan K. Armstrong PhD
Received: 17 June 2007/Accepted: 29 October 2007
? The Association of Bone and Joint Surgeons 2008
soluble polymer bone hemostatic material in a contami-
nated environment were assessed in a rabbit tibial defect
model. Infection rates and healing of polymer-treated bone
were compared with the infection and healing of bone wax-
treated bone and untreated controls after a bacterial chal-
lenge. Defects created in 24 rabbit tibias were treated with
the polymer or bone wax, or left without a hemostatic
agent. The defects were inoculated with Staphylococcus
aureus ATCC-29213 (2.5 · 104colony-forming units).
After 4 weeks, all defects treated with bone wax were
infected and osteomyelitis had developed, and none had
evidence of bone healing. In the polymer and control
groups, two defects in each group (25%) had osteomyelitis
The effects of using a newly available water-
develop. The remaining six defects in each group (75%)
showed no osteomyelitis and exhibited normal bone heal-
ing. The polymer-treated defects had a considerably lower
rate of osteomyelitis and positive bone cultures compared
with the bone wax-treated group. There were no differ-
ences between the polymer-treated and control groups in
the rates of osteomyelitis, positive cultures, or bone heal-
ing. The use of a soluble polymer as an alternative to bone
wax may decrease the rates of postoperative bone
Bone wax, which largely is composed of beeswax, is
widely used for bone hemostasis. Unadulterated beeswax
was used for amputation hemostasis during the US Civil
War. The development of modern bone wax has been
attributed to Horsley in 1892 [18, 30]. Currently available
formulations of bone wax have not changed much and are
comprised of water-insoluble beeswax softened with par-
affin and/or isopropyl palmitate . Bone wax has no
inherent hemostasis quality; its effect is to tamponade the
vascular spaces in the bone. Although effective in stopping
bone bleeding, bone wax has numerous troublesome
adverse effects . Once applied to bone, bone wax
remains at the site indefinitely. Bone wax is known to
increase infection rates, interfere with bone healing, and
elicit chronic inflammatory reactions . Continued use
of bone wax for bone hemostasis, despite its known
adverse effects, may be partly the result of the absence of a
A new synthetic bone hemostasis material made of
water-soluble alkylene oxide copolymers recently became
commercially available . The use of a water-soluble
Two of the authors (Tadeusz Wellisz, Jonathan K. Armstrong) have a
financial interest in Ceremed, Inc. Four of the authors (Yuehuei H.
An, Xuejun Wen, Qian Kang, Christopher M. Hill) received grant
support from Ceremed, Inc to support this research.
Each author certifies that his or her institution has approved the
animal protocol for this investigation and that all investigations were
conducted in conformity with ethical principles of research.
T. Wellisz (&)
Division of Plastic and Reconstructive Surgery, the Department
of Neurosurgery, University of Southern California,
536 S Rimpau Blvd, Los Angeles, CA 90020, USA
Y. H. An, X. Wen, Q. Kang, C. M. Hill
Orthopaedic Research Laboratory, Department of Orthopaedic
Surgery, Medical University of South Carolina,
Charleston, SC, USA
J. K. Armstrong
Department of Physiology and Biophysics,
Keck School of Medicine, University of Southern California,
Los Angeles, CA, USA
Clin Orthop Relat Res (2008) 466:481–486
synthetic wax for bone hemostasis comprised of similar
alkylene oxide copolymers was first described by Wang
et al. . These copolymers have a long history in the
medical and pharmaceutical fields [13, 40]. They are con-
sidered inert because they are eliminated from the body
unchanged without being metabolized [10, 16, 21].
Because these copolymers are hydrophilic, they stick well
to wet surfaces and thus are well suited for bone
Our animal study was designed to determine the
behavior of these two bone hemostasis materials in a
contaminated environment. Tibial bone defects received a
bacterial challenge after they were either treated with the
polymer material or bone wax or left untreated as a control.
The first objective of this study was to determine if the use
of the polymer material affected the infection rates; the
second objective was to determine if the polymer material
affected healing of bone defects in a contaminated
Materials and Methods
Cortical bone defects created in a rabbit tibial defect model
were treated in one of three ways. The edges of the defects
in the first group of animals were coated with a commer-
cially available blend of water-soluble alkylene oxide
copolymers (Ostene; Ceremed, Inc, Los Angeles, CA). The
U.S. Food and Drug Administration (FDA) has cleared this
material for use as an implant and for control of bleeding
from bone surfaces. The second group received a coating of
bone wax, a beeswax-based hemostat (Bone Wax; Ethicon,
Inc, Somerville, NJ). The defects in the third group were
used as controls and were not treated with a hemostatic
All procedures were approved by the Institutional Ani-
mal Care and Use Committee at the Medical University of
South Carolina. Twenty-four female New Zealand White
rabbits (2.75 ± 0.025 kg) were randomly assigned to one
of three groups (n = 8per
(0.02 mg/kg) was administered before surgery; the rabbits
were anesthetized using 30 mg/kg ketamine, 5 mg/kg
xylazine, and 1 to 3 mg/kg atropine intramuscularly and
maintained on isoflurane after intubation. Surgery was
performed using standard aseptic techniques. Animals from
each group were included at each laboratory session with
the same operators performing all surgeries. The rabbit’s
right hind limb was shaved and the skin cleaned with a
solution containing 7.5% povidone-iodine and 70% iso-
propyl alcohol. Without the use of a tourniquet, a 2.0-cm
anteromedial incision was made to access the proximal
tibia. A cortical window measuring 4 mm · 12 mm was
created at the anteromedial facet of the proximal tibia using
a 4.0-mm drill bit and a microoscillating saw under con-
stant irrigation. In the study groups, 0.25 g of material was
applied to the edges of the cortical bony defects. An
inoculum of Staphylococcus aureus strain ATCC-29230
(2.5 · 104colony-forming units in 0.1 mL saline) was
introduced into the intramedullary canal through the defect.
The organisms had been grown overnight in tryptic soy
broth at 37? C assuring confluent growth, washed twice in
phosphate-buffered saline, and resuspended in a balanced
salt solution. The plate count method was used to confirm
consistent bacterial inoculum load for all defects. The
wound was closed in layers using monofilament sutures
and the incision was covered with sterile dressing. After
surgery, the animals were given buprenorphine (0.02–
0.05 mg/kg) as needed every 12 hours.
Four weeks after surgery, the animals were euthanized
using an intravenous overdose of pentobarbital. Radio-
graphs of the tibias were taken. The tibias were exposed
through the original incisions under sterile conditions, and
bone was swabbed for bacterial culture and typing. The
tibias were harvested and cut into two segments using an
oscillating saw through the center of the original cortical
defect. The upper part of the bone explant was cultured.
Cultures were grown overnight in 5 mL of tryptic soy
broth media. A 1-mL aliquot of the culture was removed,
centrifuged to remove the growth medium, and diluted by
106with phosphate-buffered saline. One milliliter of the
diluted culture was plated on standard agar plates and
colonies were counted after 24 hours. Growth was graded
as follows: no growth; less than 20 colonies were graded
light growth; 20 to 80 colonies were graded moderate
growth; and more than 80 colonies were graded heavy
The lower part of the tibial bone and surrounding soft
tissue were fixed in 10% buffered formalin, decalcified,
and processed for paraffin sectioning. Sections were
examined microscopically after hematoxylin and eosin
staining. Image scanning of sections was performed using a
ScanScope XT System (Aperio Technologies Inc, Vista,
CA) at ·20 magnification courtesy of the Tissue Procure-
ment Core Laboratory (UCLA School of Medicine, Los
Angeles, CA). All radiographs and sections were viewed
independently by two observers (TW, XW) who were
blinded to the results.
For the infection and bone healing research questions,
the study had three treatment arms: polymer, bone wax,
and control. The results for the first question were cate-
gorized as either infected or not infected. The results for
the second question were categorized as either healing or
not healing. Statistical analysis of the data was performed
on the 3 · 2 contingency table using the Fisher-Freeman-
Halton exact test . A value of p\.05 was considered
482 Wellisz et al. Clinical Orthopaedics and Related Research
Animals that received the water-soluble polymer and ani-
mals in the control group showed a significantly lower
incidence of osteomyelitis (p £ 0.004), and positive bone
cultures (p £ 0.02), compared with the bone wax-treated
group (Fig. 1). The polymer had no effect on the infection
rate and rate of positive cultures compared with controls
(p £ 0.001). At 4 weeks, all of the animals in the bone wax
group (eight of eight) had radiographic evidence of mod-
erate to severe osteomyelitis, including periosteal reaction
and bone lysis (Fig. 2). On histologic examination, all of
the bone wax group specimens exhibited typical signs of
bone infection: development of abscess lesions, destruction
of cortical bone, and periosteal reaction. The bone marrow
structure was destroyed in all those specimens (Fig. 3). By
comparison, six of the eight animals had normal radio-
graphs in the polymer (Fig. 2B) and the control groups
(Fig. 2C); the other two animals in each group had radio-
graphic evidence of osteomyelitis together with typical
histologic signs of bone infection, including abscesses, and
destruction of bone and marrow structures.
Cultures of the swabs and bone segments were positive
for the inoculated strain of Staphylococcus aureus in 100%
of the bone wax-treated specimens. In the polymer group
and the control group, cultures from the animals with
radiographic evidence of osteomyelitis were positive, and
one additional animal of the six with normal radiographs in
each group had a positive culture; five animals in each
group had no evidence of infection.
The use of the water-soluble polymer did not affect bone
healing compared with controls (p £ 0.001). All of the
cortical defects in the animals without radiographic
evidence of infection had histologic evidence of bone
healing. In the polymer group, five of the cortical defects
had been closed by new bone formation (Fig. 4) and one
was partially closed. In the control group, four of cortical
defects were closed (Fig. 5) and two were partially
closed. None of the defects with radiographic verification
of infection had evidence of healing in any treatment
The rabbit tibial model has been used to study proposed
treatments for osteomyelitis  and provides a practical
means to investigate whether the use of a water-soluble
bone hemostasis material in a contaminated environment
might be less likely to promote the development of oste-
omyelitis than bone wax, and secondarily whether the
polymer material might influence bone healing. Some
limitations of this study are that, like with most animal
studies, there is no certainty that the findings are predictive
of the likely outcome in a human subject. Also, the type of
bacteria and the method of application do not necessarily
reflect the typical clinical situation.
In several animal studies, bone wax was shown to
increase infection rates and impair the ability of bone to
clear bacteria [22, 28, 31]. In a rabbit study, the cancellous
bone of the iliac crest was inoculated with Staphylococcus
aureus followed by placement of either bone wax or a steel
rod. The authors concluded that bone wax impaired the
ability of cancellous bone to clear the infection . In a
rat tibia model, the presence of bone wax reduced the
amount of bacteria needed to produce Staphylococcus
aureus osteomyelitis by a factor of 10,000 . In a ret-
rospective clinical study, infection rates after spinal surgery
were assessed during a 3-month period . Surgical site
infections occurred in six of 42 cases (14.3%) in which
bone wax was used and in only one of 72 cases (1.4%) in
which it was not used.
There have been no clinical reports or in vivo studies
published to date reporting complications or infections
with the use of the polymer material evaluated in this
study. One in vitro study involving one of the component
polymers (poloxamer 188) showed coating silicone wafers
with the polymer reduced bacterial adhesion and was more
effective than iodine in reducing Staphylococcus epide-
rmidis colony counts on silicone surfaces . In our
study, the use of the polymer material considerably reduced
the infection rate compared with the use of bone wax, and
it had no effect on the infection rate compared with the
The propensity to interfere with bone healing is a well-
known property of bone wax . In the 1924 edition of
Fig. 1 The application of the water-soluble polymer to a cortical
defect significantly decreased the rate of osteomyelitis formation
(p £ 0.004) and rates of positive cultures (p £ 0.02) compared with
bone wax. There was no difference between the polymer group and
the untreated control group in the rates of osteomyelitis, positive
cultures, or healing of bone defects (p £ 0.001).
Volume 466, Number 2, February 2008Infection With Bone Wax and a Soluble Polymer 483
Carson’s Modern Operative Surgery, the use of bone wax
is recommended not for bone hemostasis, but to prevent
bone healing and to create a pseudarthrosis as part of an
arthroplasty . In various animal studies, bone wax
subsequently was shown to inhibit osteogenesis and pre-
vent bone union [2, 5, 8, 11, 12, 14, 19, 20, 29, 32, 36, 40].
Bone wax remains as a foreign body at the site of appli-
cation indefinitely, and it is known to cause intense foreign
body reactions characterized by giant cells, plasma cells,
and fibrous tissue [3, 22, 28, 33, 34]. Similar findings also
were reported in humans [8, 35, 37]. Bone wax is believed
to interfere with osteogenesis, and osteoblasts have been
shown to be absent in the presence of a thin layer of bone
wax . Suggested appropriate uses for bone wax are
prevention of osteosynthesis and osteophyte formation
Fig. 2A–C (A) A radiograph taken 4 weeks after surgery shows a
bone wax-treated tibia inoculated with Staphylococcus aureus. Clear
signs of osteomyelitis can be seen, including bone lysis and periosteal
reaction. Bone explants cultured in 5% TSB showed heavy growth of
Staphylococcus aureus. (B) A representative radiograph taken 4
weeks after surgery of a polymer-treated tibia shows no evidence of
osteomyelitis and normally healing bone. (C) A representative
radiograph of an untreated control tibia also shows no evidence of
osteomyelitis and normally healing bone.
Fig. 3 A cross section of a bone wax-treated tibia at the center of the
cortical window shows typical signs of osteomyelitis, including the
development of an abscess, the destruction of cortical bone, and
periosteal reaction. The cortical window shows no signs of bone
healing after 4 weeks (arrow) (Stain, hematoxylin and eosin; original
Fig. 4 A cross section of a polymer-treated tibia at the center of the
cortical window shows typical normal bone and bone marrow without
periosteal reaction. The cortical window is filled with new bone after
4 weeks (arrow) (Stain, hematoxylin and eosin; original magnifica-
484 Wellisz et al. Clinical Orthopaedics and Related Research
The inflammatory reactions to bone wax may be a
source of postoperative pain. One report described seven
patients with intractable pain after the use of bone wax in
foot surgery . Five of the patients were pain-free after
the bone containing the inflamed bone wax was resected.
Clinical reports describing adverse inflammatory reactions
to bone wax are common [3–7, 9, 17, 23, 24, 26, 38].
Reactions consist mainly of pain and swelling, often
exacerbated by infection.
The alkylene oxide copolymer material used by Wang
et al. showed new bone grew within 10 days into a rat femur
defect with the polymer and the untreated controls . In
contrast, the defects filled with bone wax showed no bone
formation 48 days after implantation. The polymer material
dissolved from the site of application within 24 to 48 hours,
allowing the early phases of bone healing to occur . The
polymer material used in our study dissolves in the body
and has been shown not to interfere with bone healing or
cause inflammation in a sterile environment .
In this study, in the presence of bacterial contamination,
the use of the polymer material neither increased infection
rates nor interfered with bone healing when compared with
untreated controls. All of the defects without radiologic
evidence of osteomyelitis had normal bone healing. The
use of this polymer material in place of bone wax may be
another step toward reducing wound complications and the
associated morbidity after bone surgery.
Tissue Procurement Core Laboratory, Department of Pathology and
Laboratory Medicine, UCLA School of Medicine, Los Angeles, CA,
for providing the microscopic images of hematoxylin and eosin-
We thank Drs S. Dry and D. Gui from the
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