Infected open fractures result in substantial morbidity, especially
on the modern battlefield, and the hospital stay is doubled for patients in
whom infection
develops1. To help
prevent infection in an open fracture, a well-established treatment has been
the local delivery of antibiotics with use of polymethylmethacrylate beads
impregnated with the
drug2-4.
Local delivery of antibiotics has the advantage of providing high local
concentrations with low serum concentrations, thereby avoiding systemic
toxicities3.
However, cement beads require surgical removal because antibiotic elution
decreases by two to six weeks, and the beads become nothing but a foreign
body2. In addition,
polymethylmethacrylate itself does nothing to assist healing.
An ideal antibiotic delivery system would enhance healing, elute the entire
antibiotic dose, and require no additional surgery for bead removal. Numerous
animal studies have demonstrated that biodegradable local antibiotic delivery
systems are safe and effective for treatment of
osteomyelitis5-12.
Recently, McKee et
al.13 and Gitelis
and Brebach14
showed that tobramycin-impregnated calcium sulfate pellets (OSTEOSET T) are
effective in treating chronic osteomyelitis in humans.
Calcium sulfate has been used since 1892 as a bone defect filler and is
known to be
osteoconductive15,16.
Similarly, demineralized bone matrix is known to be
osteoinductive15.
Turner et al. showed, in a canine model, that the combination of calcium
sulfate pellets and demineralized bone matrix is more effective as a
bone-graft substitute than is either calcium sulfate or demineralized bone
matrix alone17.
Additionally, they demonstrated that the combination was as effective as
autogenous bone graft at six weeks following treatment. Therefore, we
hypothesized that the combination of tobramycin-impregnated sulfate pellets
and demineralized bone matrix could be used as a local antibiotic delivery
system that is osteoinductive and osteoconductive as well as
antimicrobial.
The purpose of this study was to evaluate the efficacy of a locally
delivered antimicrobial with bone-graft substitute in the prophylactic
treatment of infection in an open fracture. We hypothesized that the
combination of tobramycin-impregnated calcium sulfate pellets and
demineralized bone matrix would prevent the establishment of infection in a
contaminated fracture model.
Animal Handling
The Institutional Animal Care and Use Committee approved all
experiments and animal care procedures. All procedures were conducted in an
animal facility approved by the Association for Assessment and Accreditation
of Laboratory Animal Care, and all were performed in accordance with the
National Institutes of Health guidelines for care and use of laboratory
animals.
Forty-eight Spanish goats with a weight range of 37 to 50 kg (mean and
standard deviation, 42 ± 4 kg) were initially enrolled in the study;
sample-size estimation with 81% power identified a requirement for twelve
animals per group for the main experimental study.
All goats were housed in runs in a climate-controlled facility and were fed
commercial food and water ad libitum. The goats were tested for tuberculosis,
brucellosis, and Q fever, and they were observed for ten to fourteen days
prior to the study to allow for environmental changes and to exclude the
possibility of preexisting disease. A veterinarian examined each goat prior to
commencement of the protocol.
Surgical Technique
The goats were not fed for forty-eight hours prior to the surgery, and
water was withheld for twelve hours. After adequate regional and general
anesthesia was achieved, the right lower extremity was prepared with
chlorhexidine gluconate and draped in a sterile fashion. To avoid confounding
variables, no preoperative intravenous antibiotics were given. A 2.5-cm
longitudinal skin incision was made over the medial aspect of the proximal
metaphyseal region of the tibia, centered at a point approximately 2 cm medial
and 2 cm distal to the tibial tubercle. After the periosteum was elevated, a
unicortical, 12-mm circular defect was produced with a coring reamer. A
thrombin-soaked gelatin sponge was used to assist in medullary hemostasis. The
osseous defect was inoculated with an aliquot of bacteria (30 µL of
solution with a mean of 3.14 × 106 CFU/mL of
Staphylococcus aureus). Thirty microliters of a 106 CFU/mL
solution of bacteria has been shown to be sufficient to cause infection
without producing sepsis in >70% of nontreated
animals18. The
bacterial strain used was American Type Culture Collection (ATCC) 29213
(Manassas, Virginia), which was further modified by our institution to be
resistant to streptomycin. Aliquots of Staphylococcus aureus were
plated on streptomycin plates. The isolates with the highest concentration
were recovered and were grown overnight in brain-heart infusion broth. This
process was repeated twice more, and after the third pass the mutant was
streaked on plates containing various concentrations of streptomycin (50 to
1000 µL/mL) to ensure resistance.
Before the study was started, the forty-eight goats were randomized into
four groups of twelve animals each. A preplanned interim statistical analysis,
which our institution's animal care committee had requested to conserve
animals if statistical significance was achieved, showed that significance had
been achieved midway through data collection. Thus the study was halted,
resulting in the groups containing six, seven, or eight goats. The negative
control group (seven goats) received no treatment. In the positive control
group (six goats), fifteen to nineteen handmade tobramycin-impregnated
polymethylmethacrylate beads (Palacos; Biomet, Warsaw, Indiana), resulting in
a mean dose of 113.5 ± 7.3 mg of tobramycin sulfate, were used to fill
the bone defect of each goat. The inconsistent dose and number of beads in
this group was due to the slight variation in the sizes of the handmade beads
as well as the need to adjust the numbers of beads to fill the metaphyseal
voids of differently sized goats. The demineralized bone group (eight goats)
had 2.5 mL of demineralized bone matrix (Allomatrix injectable putty; Wright
Medical, Arlington, Tennessee) placed into the metaphyseal defect. The
experimental group (eight goats) received fifteen pellets of 10%
tobramycin-impregnated calcium sulfate (OSTEOSET T; Wright Medical) with 2.5
mL of demineralized bone matrix. The fifteen pellets of OSTEOSET T had a total
of 160 mg of tobramycin sulfate.
Wound Evaluation
The animals were followed daily for twenty-one days for clinical signs of
infection. Abscess formation was followed clinically by measuring leg
circumference. Abscesses that increased in size over three days were relieved
with aspiration or incision and drainage. Discharge from abscesses was sent
for culture and bacterial identification. At the first sign of wound drainage,
the discharge was also cultured, and bacterial species were identified. All
Staphylococcus aureus isolates were tested for streptomycin
resistance to determine if the cultured strain was identical to the original
bacterial inoculate. If any animal demonstrated discomfort or distress during
the three-week observation period, a 100-µg/hr fentanyl citrate patch was
placed on the skin of the neck area and secured with elastic bandaging tape
under the supervision of a veterinarian. On postoperative day 21, all of the
goats were killed and necropsy studies were performed as described below.
Wound Grading System
Clinical signs of wound infection included erythema, inflammation, and
purulent drainage. After the dressing was removed on postoperative day 4,
three independent examiners graded each wound daily. The graders were blinded
to the treatment groups for the duration of the study. The clinical grading
system that was used had been established in a previous caprine
study18. The wound
was assigned a score of 0 when there were no signs of contamination or
swelling; a score of 1 when there was inflammation, swelling, or serous
drainage without frank purulence; and a score of 2 when there was frank
purulence at the wound site or purulent discharge on aspiration or incision
and drainage. A total score for each wound was calculated by adding the scores
assigned by each of the three observers each day for twenty-one days. The
clinical determination of infection was defined by a score of at least 4 on
two consecutive days. Hence, a wound had to exhibit purulent drainage, as
identified by two of the three examiners, for two consecutive days to be
considered infected.
Necropsy and Microbiologic Analysis
On postoperative day 21, the goats were killed, the treated hindlimb was
disarticulated at the hip, and anteroposterior and lateral radiographs were
made. Soft tissue was removed from the tibia, and the osseous defect was
transected at its midportion with a Gigli saw. Culture swabs were obtained by
swabbing the canal proximal and distal to the defect. A number-5 surgical
curet (to obtain 0.5 g of tissue) was used to harvest marrow and trabecular
tissue from the canal. Finally, a 2-cm portion of the tibia encompassing the
osseous defect was removed with a sagittal saw.
The tissue and swab samples were sent for culture and identification of the
bacterial species. Each Staphylococcus aureus isolate was tested for
streptomycin resistance to determine whether it was the same strain as the
initial inoculum.
Outcome Measure
The outcome measure used to identify a deep wound infection was the
recovery of the streptomycin-resistant Staphylococcus aureus strain
ATCC 29213 from intramedullary cultures at twenty-one days. The threshold for
infection was set at 104 CFU/g of
marrow18. Cultures
in which bacteria were present but the count was <103 CFU/g of
marrow were considered to be contaminated. If the quantitative analysis
identified a bacteria count of between 103 and 104 CFU/g
of marrow in the final tissue culture, the clinical score was used to
determine whether an infection was present (i.e., the wound had to be
considered infected by our clinical scoring criteria to be considered
infected).
Statistical Analysis
Analysis of variance was used to ensure that the groups were comparable in
terms of body weight and amount of inoculation (CFU/g of marrow). A
nonparametric median test was used to test for differences between treatment
groups. Clinical scores were compared between groups with use of a chi-square
test. When global differences were detected, a Bonferroni adjustment was used
for error correction in order to determine the significance of subsequent
comparisons.
None of the animals displayed signs of systemic sepsis (decreased
activity or alertness, lack of appetite, or fever). There was no significant
difference between groups with respect to the mean body weight of the goats (p
= 0.96) or the mean amount of bacterial inoculum (p = 0.86).
In the negative control group, the wound scores ranged from 0 to 6 during
the clinical evaluation period. Purulent drainage developed in one goat, which
received a wound score of 6. Four goats received a wound score of 3; one goat,
with negative cultures, received a score of 0 (normal); and another goat, with
positive cultures, received a score of 0. (see Appendix). All of the goats in
this group showed evidence of periosteal reaction on the final radiographs.
Gross pathologic examination demonstrated necrosis and abscess formation in
five of the seven goats. Cultures at twenty-one days confirmed intramedullary
infection with streptomycin-resistant Staphylococcus aureus in six of
the seven goats. The mean count in the group was 2.2 × 108
± 3.3 × 108 CFU/g
(Table I).
In the positive control group, all six goats had wound-healing without any
signs of infection during the clinical evaluation period, and all received a
wound score of 0. None of the final radiographs showed evidence of periosteal
reaction, and gross pathologic examination showed no signs of infection.
Cultures did not demonstrate bacteria in the intramedullary tissues of any of
the goats in this group.
In the demineralized bone matrix group, purulent discharge developed, and
the wound score was 6, in six of the eight goats. All eight goats showed
evidence of periosteal reaction on radiographic examination. Gross pathologic
analysis demonstrated abscesses, necrosis, and draining sinuses in seven of
the eight goats. Cultures at twenty-one days confirmed intramedullary
infection with streptomycin-resistant Staphylococcus aureus in seven
of the eight goats. The mean count was 1.3 × 108 ± 2.3
× 108 CFU/g (Table
I). One goat had purulent discharge that was positive for
streptomycin-resistant Staphylococcus aureus on culture during the
clinical evaluation period, but the final culture demonstrated
Streptococcus viridans in the intramedullary tissue.
In the experimental group, four of the eight goats had early,
culture-negative, serous discharge for two to six days during the clinical
evaluation period. Two goats in this group had superficial wound infections;
one was infected with non-hemolytic Streptococci, and one infection resolved
after removal of a small superficial eschar. Radiographic examination revealed
periosteal elevation in six of the eight goats; however, the degree of
reaction was qualitatively less than the reaction seen in both the negative
control and the demineralized bone matrix group. Gross pathologic examination
demonstrated incorporation of the demineralized bone matrix and calcium
sulfate pellets without evidence of infection
(Fig. 1). There was no growth
of any bacteria on culture of the intramedullary tissues from the eight goats
in this group.
All goats in both groups that had received tobramycin had negative cultures
for intramedullary bacteria at twenty-one days
(Table I). There were
significant differences between the treatment groups in terms of the number of
CFU/g (p < 0.0001). The positive control group had a significantly lower
number of CFU/g than the negative control group (p = 0.018) and the
demineralized bone matrix group (p = 0.011). Similarly, the experimental group
showed a significantly lower number of CFU/g than either the negative control
(p = 0.0066) or the demineralized bone matrix group (p = 0.0006). With the
numbers available, the negative control and demineralized bone matrix groups
were not significantly different from one another in this regard (p =
0.46).
The demineralized bone matrix group had a significantly higher number of
clinical infections (a score of at least 5 on two consecutive days) than did
the experimental group (p = 0.006), the positive control group (p = 0.01), or
the negative control group (p = 0.02). There was no significant difference in
the wound score between the experimental group and the positive control group
(p = 0.09).
The current study demonstrated that an antibiotic-impregnated
bone-graft substitute can be used effectively to treat infection in a
contaminated fracture model. Previous animal studies and human clinical trials
have shown tobramycin-impregnated calcium sulfate pellets to be effective in
the treatment of
osteomyelitis7,13,14,19.
Since biodegradable antibiotic drug delivery systems are effective against
osteomyelitis, it stands to reason that they might also be effective as
prophylaxis. Previous work at our institution showed that
tobramycin-impregnated calcium sulfate pellets by themselves provided
effective prophylaxis in a similar goat
model20. However,
calcium sulfate is only osteoconductive, and, in a canine model, it took
twenty-four weeks for it to stimulate as much bone growth as was produced in
defects treated with autogenous bone
graft17. The
combination of calcium sulfate pellets and demineralized bone matrix is
osteoconductive and osteoinductive, and it stimulated as much bone growth as
did autograft in just six weeks in the same
model17. We
hypothesized that, with the addition of tobramycin, the combination should be
antimicrobial as well, but this combination had never been tested in a
contaminated fracture model, to our knowledge.
In the contaminated fracture model used in this study, the combination of
tobramycin-impregnated calcium sulfate pellets and demineralized bone matrix
did not differ significantly from tobramycin-impregnated cement beads with
regard to its ability to prevent the establishment of Staphylococcus
aureus infection. Therefore, this combination could decrease the
morbidity of open fractures by eliminating the need for removal of surgical
beads and by reducing the number of autografts harvested.
One limitation of this study was that the degree of bone-healing was not
compared between groups. This model was developed to determine the ability of
a treatment to prevent an infection in a contaminated bone defect, which is a
necessary first step in assessing the usefulness of a treatment for an open
fracture. However, the ability of this combination to promote healing of open
fractures is, as yet, untested. Another limitation of this study is that the
experimental group received more tobramycin sulfate than did the positive
control group because the calcium sulfate pellets had a higher concentration
of tobramycin sulfate than did the handmade beads (10% compared with 4% by
weight). Handmade beads are the current standard of care, and the current
recommendation is to mix 2.4 g of tobramycin with 40 g of
polymethylmethacrylate, which produces the above
concentration21.
This was not intended to be a definitive dose-response study; still, the
higher concentration and load of tobramycin that the experimental group
received may have biased that group's success. However, this concentration
does not adversely increase serum tobramycin
levels17.
An important observation was the serous drainage that occurred for two to
six days in the experimental group. Similar drainage has been observed in
clinical trials, in which the investigators noted that the drainage halted
when there was radiographic evidence of resorption of the calcium sulfate
pellets7,13,14,19.
This drainage may explain the two superficial wound infections in the
experimental group. Additional study will focus on the effect of this
combination on bone-healing and compare this approach with other current
standards of care such as the use of antibiotic cement beads in combination
with intravenous antibiotics.
A table presenting the wound scores and culture results for all study
animals is available with the electronic versions of this article, on our web
site at
(go to the article citation and click on "Supplementary Material")
and on our quarterly CD-ROM (call our subscription department, at
781-449-9780, to order the CD-ROM). ?