Ultrasound is currently applied for diagnostic and therapeutic
purposes1.
Ultrasonic intensities for therapeutic and surgical procedures are high (1 to
50 W/cm2) and can cause considerable
tissue-heating2. In
contrast, the intensity for diagnostic procedures is low (1 to 50
mW/cm2) and is considered to be nonthermal and
safe3,4.
To our knowledge, low-intensity ultrasound was first used to enhance
fracture-healing by
Duarte5, who
reported good results. The positive effect of low-intensity ultrasound on the
rate of osseous repair has been well documented in several
animal6-9
and
clinical10-12
studies.
Animal studies have demonstrated that ultrasound treatment substantially
increases the strength of a healing fracture. Pilla et
al.7 reported that
the mechanical properties of a healing rabbit fibular fracture were
accelerated by a factor of nearly 1.7 after application of low-intensity
ultrasound. Wang et
al.8 as well as Yang
et al.9 used a rat
bilateral femoral fracture model with application of ultrasound to one of the
two fractures. Both studies showed that the average maximum torque and
torsional stiffness were significantly greater in the ultrasound-treated limbs
than in the control limbs.
The ultrasound intensity level in most reported series has ranged from 30
to 100 mW/cm2—in the range of intensities used for diagnostic
imaging—in order to avoid harmful effects. In most series, the fracture
sites were exposed to ultrasound for twenty minutes per day. In the
above-mentioned studies, the mechanical energy of the ultrasound was
transferred to the tissues through a transducer, which was applied to the skin
over the site of the fracture. The same technique was used in clinical
studies11,12.
However, ultrasonic energy has to pass through skin, fat, muscle, and hematoma
before reaching the fracture site, and there is absorption of the ultrasonic
energy as the ultrasound wave is transmitted through these tissues.
Our hypothesis was that application of the ultrasound directly to bone,
bypassing the overlying tissues, would be a feasible, safe, and effective way
to enhance bone-healing. The goals of our study were (1) to develop a novel,
transosseous method for ultrasound transmission, and (2) to test the safety
and efficacy of this method in accelerating fracture-healing.
Experimental Design and Animal Model
Forty skeletally mature sheep weighing 45 to 55 kg were randomly assigned
to two groups of equal size: the ultrasound group and the control group. An
osteotomy was created in the tibia of the left hindlimb and was stabilized
with an external fixator. Transosseous ultrasound was applied to the osteotomy
site in the twenty animals in the ultrasound group, whereas no other
intervention was performed in the twenty animals in the control group.
Ten sheep from each group were killed on postoperative day 75, and ten were
killed on postoperative day 120. Both the operatively treated left tibia and
the contralateral tibia were harvested, and quantitative computed tomography
and subsequent biomechanical testing were performed.
The research protocol was reviewed and approved by the Animal Committee of
the University of Ioannina. All animals appeared to be healthy and were housed
individually with appropriate husbandry conditions before and after the
procedure.
Transosseous Ultrasound Transmission Model
Transosseous ultrasound transmission was performed with a method that was
developed for the current investigation. A custom-made stainless-steel pin,
measuring 40 mm in length and 2 mm in diameter, with one sharp and one blunt
end, was used. The blunt end was specially designed to accept and firmly hold
a PZT-4D transducer (Morgan Matroc, Bedford, Ohio) in place to avoid
dislodgment during movement of the animals. This pin-transducer apparatus
enables the ultrasound energy to be transmitted through the free end of the
pin to the sharp end, which is inserted 1.0 cm proximal to the osteotomy
site.
The pin-transducer apparatus was tested at the Ultrasound Laboratory of the
Department of Physics of the University of Ioannina to ensure that no
substantial loss of ultrasound energy occurred at the connection site or
during propagation of the ultrasound wave through the pin. The 1-cm distance
of the pin from the osteotomy site was selected to maximize the energy
absorbed at the osteotomy site.
Operative Procedure and Ultrasound Application
Anesthesia was achieved with use of ketamine (10 mg/kg) and xylazine (3
mg/kg). The hindlimbs were shaved, scrubbed with Betadine (povidone-iodine,
10% solution), and draped. Under aseptic conditions, a Monotube unilateral
external fixator (Howmedica Jaquet, Geneva, Switzerland) was mounted on the
anterolateral aspect of the left tibia with use of a four-pin technique. The
tibia was exposed subperiosteally, and a mid-diaphyseal osteotomy was
performed, starting with multiple adjacent 2.0-mm drill holes and completing
the osteotomy with a bone chisel.
The custom-made pin was applied 1 cm proximal to the osteotomy site and
perpendicular to the anterolateral aspect of the cortex in all animals. The
pin was inserted into the medullary canal, and it engaged the opposite cortex
for additional stability. A second pin was also inserted, 1 cm distal to the
osteotomy site, but this was used only to monitor fracture-healing with
ultrasound in both groups. In the treatment group, ultrasound energy was
transmitted through the free end of the proximal pin with a PZT-4D transducer.
Radiographs were used to make certain that the osteotomy was transverse and
not displaced (Fig. 1). The
skin and fascia were closed with absorbable sutures.
The sheep were allowed unrestricted walking in their cages after they
recovered from the anesthesia, and they were observed daily for activity,
wound-healing, and development of pin-track infection.
The animals in the treatment group received ultrasound exposure for twenty
minutes daily, starting on the first postoperative day, until they were
killed. The ultrasound signal consisted of a 200-µsec burst sine wave of
1.0 MHz repeating at 1.0 kHz. The intensity was 30 mW/cm2 spatial
average and temporal average.
Radiographic Assessment of Healing
Anteroposterior and lateral radiographs were made every fifteen days for
all animals. However, only the radiographs of animals that were killed on
postoperative day 120 were used for the radiographic assessment of healing, in
order to allow adequate time for healing in both groups.
We used the method of Heckman et
al.11 for
radiographic assessment of healing. At each time-point, four cortices (two on
the anteroposterior radiograph and two on the lateral radiograph) were
evaluated for cortical bridging. All radiographs were assessed independently
by a radiologist (A.H.K.) and an orthopaedic surgeon (C.G.Z.), who were
blinded to the treatment group. Radiographic healing was defined as the time
at which three of the four cortices were bridged.
Specimen Harvest
At the predetermined time-points, the sheep were killed, after induction of
general anesthesia with sodium pentobarbital, with intravenous administration
of potassium chloride. Soft tissues were dissected from both tibiae of each
animal, and the denuded tibiae were stored in 70% ethanol for the purposes of
quantitative computed tomography scanning and mechanical testing.
Quantitative Computed Tomography and Biomechanical Testing
Quantitative computed tomography (Philips LX Plus; Philips Medical Systems,
Best, The Netherlands) was performed at the level of the osteotomy. At each
cross section, the density of three regions in the cortical zone was measured
(Fig. 2), and the average of
the three cortical values was described as cortical bone mineral density. For
mechanical testing, all specimens were tested in a computerized testing
machine (Karl-Frank, Bern, Switzerland) applying a three-point bending test at
room temperature13.
All bones were loaded with a low strain rate (0.05 mm/sec) until failure, and
ultimate strength (N) and stiffness (N/mm) were determined from the
load-displacement curve. The bending load was applied to the specimen at the
site of the osteotomy and in the sagittal plane. All specimens were fixed on
the three-point bending apparatus, and the span of loaded bone was 8 cm to
guarantee that 90% of the flexion of the bone was due to bending and not to
torsion13.
Statistical Analysis
The Mann-Whitney two-tailed nonparametric test was used to compare the
median times to healing between the two groups. Analysis of variance was used
to assess densitometric and biomechanical data, followed by the Tukey test for
pairwise comparison between groups. Significance was accepted at p <
0.05.
All animals survived until the end of the study. A minor external-fixation
pin-track infection developed in five animals (three from the ultrasound group
and two from the control group), and all were easily treated with local care
of the pin track. No pin had to be removed, and osteomyelitis did not develop
in any of the animals. No infection was noted at the sites of the pins used
for ultrasound transmission.
There was a significant difference between the ultrasound and control
groups with regard to cortical bone mineral density at seventy-five days
postoperatively. The mean cortical bone mineral density (and standard
deviation) was 781 ± 52 mg/mL in the treatment group and 543 ±
44 mg/mL in the control group (p = 0.014, analysis of variance)
(Table I). At seventy-five
days, central callus resorption and remodeling of the periosteal callus had
started in the treatment group. In contrast, the callus in the control group
covered the entire cross section, including the marrow canal, without
remodeling of the cortex area (Figs.
3-A and
3-B). However, at 120 days, the
difference between the treatment and control groups was not significant (941
± 81 mg/mL and 906 ± 69 mg/mL, respectively; p = 0.78, analysis
of variance) (Table I).
Mechanical testing with the lateral bending test on postoperative day 75
revealed significantly greater ultimate strength and stiffness in the treated
limbs compared with the control limbs. The average ultimate strength was 1928
± 167 N in the treatment group and 1493 ± 112 N in the control
group (p = 0.012, analysis of variance)
(Table II). Nevertheless, the
ultrasound-treated limbs as well as the control limbs were significantly
weaker than the intact tibiae (p = 0.001) at that time. As was the case with
the cortical bone mineral density, by postoperative day 120 the difference in
ultimate strength between the treatment and control groups was not significant
(2361 ± 154 N and 2286 ± 135 N, respectively; p = 0.29, analysis
of variance). Similarly, the stiffness in the treated limbs was significantly
greater than that in the control limbs on postoperative day 75 (p = 0.034),
but there was no significant difference between the groups on postoperative
day 120 (Table III).
The radiographs of the ten animals from the treatment group and the ten
from the control group that were killed on postoperative day 120 were analyzed
to assess healing time. Although all fractures healed by 120 days in both
groups, there was a significant difference between the two groups with respect
to the time of healing. The median time for radiographic healing (bridging of
three of the four cortices), as assessed by the orthopaedic surgeon, was
seventy-nine days (range, sixty to ninety days) for the treatment group and
103 days (range, seventy-five to 105 days) for the control group (p = 0.027,
Mann-Whitney test). As assessed by the radiologist, on review of the same
radiographs, the corresponding times were eighty-eight days (range, sixty to
105 days) compared with 109 days (range, seventy-five to 120 days) (p = 0.038,
Mann-Whitney test) (Figs. 4-A,
4-B,
5-A, 5-B).
Many basic
science5-9
and clinical
studies10-12
have shown that low-intensity ultrasound can accelerate and augment the
fracture-healing process. The mechanisms by which low-intensity ultrasound
interacts with tissue and accelerates fracture-healing remain unknown. It has
been hypothesized that when ultrasound passes through tissues, absorption of
ultrasonic energy occurs at a rate proportional to tissue
density14. This
energy absorption has been reported to increase cell metabolic
activity3, stimulate
vascular
activity14,15,
increase gene
expression9,16,
enhance calcium incorporation at the fracture
site17, offer a
beneficial mechanical stimulus in the healing
callus18, and
enhance fracture-healing.
At interfaces of tissues with different densities, such as at bone-callus
surfaces, much of the incident radiation energy is reflected, resulting in
complex gradients of acoustic pressure through the tissue and reduction of the
transmitted
energy19. Because
of this, we were motivated to investigate the feasibility of direct
transmission of ultrasound through bone to the callus area and to determine
whether the transosseous method could be an alternative to the conventional
transcutaneous technique to promote fracture-healing. To our knowledge, we are
the first to investigate the effect of transosseous low-intensity ultrasound
on fracture-healing.
Our custom-made apparatus was able to transmit ultrasound waves directly to
the callus area, which enhanced the healing process. Insertion of the
apparatus pin in close proximity to the osteotomy site did not have any
adverse effects. We were concerned that the pin could be a source of
infection. Although five minor external-fixation pin-track infections
developed in the animals, they did not compromise fracture-healing.
Transosseous application of low-intensity ultrasound enhanced bone-healing
effectively in this animal model, as demonstrated by the biomechanical,
quantitative computed tomography, and radiographic data. The biomechanical
testing demonstrated that low-intensity transosseous ultrasound is capable of
significantly accelerating the biomechanical properties of a healing osteotomy
site in a controlled fracture model. On postoperative day 75, the average
stiffness of the treated fractures was 40% greater than that of the control
fractures. Previous
investigators9,20
have attributed this increase to stimulation of chondrogenesis and cartilage
hypertrophy by ultrasound exposure, resulting in an earlier onset of
endochondral bone formation and subsequently in increased fracture callus
strength in the early phases of healing. However, by postoperative day 120,
there was no significant difference between our two groups.
Analysis of the callus region with quantitative computed tomography also
revealed a significant difference between the ultrasound and control groups
with regard to cortical bone mineral density on postoperative day 75. Computed
tomography allows three-dimensional imaging and an objective estimation of the
callus region. Our analysis of the computed tomography images showed that the
stage of callus healing differed between the two groups. Reconstitution of the
marrow canal and cortical remodeling had started in the limbs treated with the
low-intensity ultrasound, whereas cancellous bone was more abundant and the
cortical remodeling was not obvious in the control limbs. Thus, acceleration
of the healing process was observed in the treated limbs. This finding is in
agreement with that of Azuma et
al.20, who found,
with densitometric methods, that low-intensity ultrasound increased bone
mineral density and accelerated the overall endochondral ossification process.
However, by postoperative day 120, we observed no significant differences
between our control and treatment groups.
To assess the rate of acceleration of fracture-healing, we used
radiographic criteria similar to those employed by Heckman et
al.11 and
Kristiansen et
al.12. We found
that transosseous low-intensity ultrasound significantly reduced the median
time to radiographic healing (from 103 days in the control group to
seventy-nine days in the ultrasound treatment group, p = 0.027) as assessed by
the orthopaedic surgeon, but all fractures in both groups had healed by
postoperative day 120.
Two double-blinded, randomized, placebo-controlled clinical
studies11,12
showed that transcutaneous low-intensity ultrasound significantly reduced the
time for radiographic healing. Heckman et
al.11 found that
ultrasound led to a 40% decrease in the time for radiographic healing of
tibial fractures treated with a cast, with the ultrasound-treated fractures
healing in eighty-nine days compared with 148 days in the control group (p
< 0.001). Similarly, in another study of the time to union of distal radial
fractures treated with a cast, Kristiansen et
al.12 found that
ultrasound shortened the time to healing by 38%.
The results of our study should be viewed with caution. We utilized a sheep
osteotomy model, which differs from the approach in the two above-mentioned
studies. The sheep model has limited applicability to human fractures, so
caution should be used in extrapolating our results to humans. Moreover, the
fact that pin-track infections were only a minor problem in sheep does not
guarantee safety in clinical practice because, in humans, the pin may need to
be in place for a longer period of time and infectious complications could
develop. As a consequence, the clinical benefits of the transosseous technique
remain to be established. The current investigation should be viewed as a
preliminary experimental study, and the results should not be applied to human
clinical practice before additional investigations address the aforementioned
reservations. However, previous studies have shown that transcutaneous
ultrasound was effective in both
animals5-9
and
humans10-12.
Another limitation of our study is that we did not compare transosseous and
transcutaneous methods. However, we wanted to first establish the feasibility,
safety, and efficacy of the transosseous method before proceeding with a
direct comparison. Our study is also limited by the lack of a histological
analysis, which could have provided information regarding canal reconstitution
and cortical remodeling. Quantitative computed tomography extraction of the
relevant morphometric parameters regarding the cancellous and cortical bone
compartments would have also been beneficial by delineating bone structure at
the healing osteotomy site.
Our study suggests that transosseous application of low-intensity
ultrasound close to the fracture site enhances the mechanical properties of
the fracture callus, increases the cortical bone mineral density of the
regenerated bone, and reduces the time to healing. Additional studies are
needed to determine whether this invasive method is advantageous in comparison
with the conventional transcutaneous method and to clarify the technical
details of optimal application.