Effects of Hyperbaric Oxygen Treatment on Tendon Graft
and Tendon-Bone Integration in Bone Tunnel: Biochemical
and Histological Analysis in Rabbits
Wen-Ling Yeh,1 Song-Shu Lin,1 Li-Jen Yuan,1 Kam-Fai Lee,2 M.Y. Lee,3 Steve W.N. Ueng1
1
Department of Orthopaedic Surgery and Hyperbaric Oxygen Therapy Center, Chang Gung Memorial Hospital,
5, Fu-Hsin St. 333, Kweishan, Taoyuan, Taiwan
2
Department of Pathology, Chang Gung Memorial Hospital, 5, Fu-Hsin St. 333, Kweishan, Taoyuan, Taiwan
3
Department of Mechanical Engineering, Chang Gung University, Taiwan
Received 28 September 2005; accepted 10 November 2006
Published online 2 February 2007 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jor.20360
ABSTRACT: Despite moderate success in clinical applications, outcome of tendon grafts employed
for anterior cruciate ligament (ACL) reconstruction remains unsatisfactory. This study investigated
the effects of hyperbaric oxygen (HBO) on neovascularization at the tendon-bone junction, collagen
fibers of the tendon graft, and the tendon graft-bony interface incorporated into the osseous tunnel in
rabbits. Forty rabbits were assigned to two groups. The HBO group was exposed to 100% oxygen at
2.5 atmospheres pressure for 2 h daily, 5 consecutive days in a week. The control group was
maintained in cages exposed to normal air. Histological studies of 12 rabbits were performed
postoperatively at 6, 12, and 18 weeks. Biomechanical studies of 24 rabbits were conducted
postoperatively at 12 and 18 weeks. Electron microscopy (EM) analyses of four rabbits were
performed postoperatively at 18 weeks. Experimental results demonstrated that a higher number of
Sharpey’s fibers bridged the newly formed fibrocartilage and graft in the HBO group than in the
control group. In addition, HBO treatment increased neovascularization and enhanced the
incorporation of the progressive interface between tendon graft and bone. Biomechanical analysis
showed that the HBO group achieved higher maximal pullout strength than the control group.
Examination by EM showed that HBO treatment resulted in regenerated collagen fibers with
increased compaction and regularity. Based on experimental results, HBO treatment is a treatment
modality that potentially improves outcome following ACL reconstruction. ß 2007 Orthopaedic
Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 25:636–645, 2007
Keywords: hyperbaric oxygen; ACL reconstruction; graft-bone interface; neovascularization; collagen fibrils
INTRODUCTION
Anterior cruciate ligament (ACL) reconstruction
using semitendinosus and gracilis tendons
achieves unsatisfactory outcomes. Successful
ACL reconstruction with a tendon graft requires
solid healing of the graft in the bone tunnels of the
femur and tibia. However, the process of tendon
graft and bone incorporation remains unclear.
Sharpey and Ellis1 described perforating fibers
that extend across the bone, anchoring the soft
tissue to the bone. The presence of these fibers
should be considered to represent an indirect
Correspondence to: Steve W.N. Ueng (Telephone: 866-33281200 ext. 3223; Fax: 886-3-3278113;
E-mail: wenneng@adm.cgmh.org.tw)
ß 2007 Orthopaedic Research Society. Published by Wiley Periodicals,
Inc.
636
JOURNAL OF ORTHOPAEDIC RESEARCH MAY 2007
tendon or ligament insertion, as can be found for
the periosteal attachment of tendons.2,3 Rodeo
et al.4 and Blickenstaft et al.5 demonstrated an
indirect tendon insertion in the osseous tunnel,
whereas Grana3 showed that semitendinosus
tendon grafts can be pulled out of the osseous
tunnel during tensile testing, leaving a fibrous
tissue lining within the tunnel. Conversely, Shino
et al.7 achieved direct tendon insertion in the
osseous tunnel in dogs after 30–52 weeks.
Improved tendon healing in the osseous tunnel
will likely result in enhanced clinical outcome for
ACL reconstruction. Biological fixation of the graft
in the osseous tunnel and the behavior of the intraarticular section of the graft as it undergoes
revascularization and maturation require investigation.6,8 During the healing process, the tendon
graft is infiltrated by inflammatory tissues and
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HBO TREATMENT ON TENDON GRAFTS IN RABBITS
is subject to ischemic change. Fibroblasts will
repopulate in the tendon graft coinciding with
neovascularization by 4 weeks after surgery and
the risk for injury to the knee joint remains
significant during this period.9 Thus, risk of ACL
failure can be minimized with improved neovascularization and shortened ischemic time. Bosch
et al.33 demonstrated that tendon graft collagen
fibrils become smaller and are more compact with
predominant type III collagen fibers after reconstruction of the posterior cruciate ligament using a
patellar tendon graft. Hyperbaric oxygen (HBO)
has been shown to enhance the angiogenesis in
various tissues,10–12 and increase healing of the
extra-articular ligament reconstruction.13 However, the effect of HBO on the intra-articular
ligament reconstruction is unknown. Because
tendon-to-bone healing occurs bony in-growth,4,20
it is possible that healing could be improved
by adding exogenous bone-growth treatments.
We hypothesized that HBO treatment increases
neovascularization at the tendon-bone junction
and tensile strength of the tendon graft, and
enhances tendon-bone incorporation. This study
employed a rabbit model to evaluate the effects of
HBO on neovascularization at the tendon-bone
junction, collagen fibers in reconstructed hamstring tendon grafts, and the tendon graft-bony
interface incorporated inside the osseous tunnel.
with 2-0 prolene sutures (J&J; Ethicon, Edinburgh,
England). The knee joint was put through 10 cycles of
motion. Maximal tension was applied to the graft with
the knee flexed at 308 before securing the graft.
The wound was closed with 3-0 nylon (J&J; Ethicon) in
the interrupted fashion. Postoperatively, ampicillin
(50 mg/kg) was administered intramuscularly for 3 days.
No special immobilization of the right hind limb was
applied and the rabbit was free to move around in the
housing case.
Histological Study and Scoring System for
Histological Analysis
Four rabbits were sacrificed at 6, 12, and 18 weeks after
operation. Specimens were fixed in 10% neutral buffered
formalin for 1–2 days, and then decalcified in nitric acid
for 7–8 days. For routine histological examination,
paraffin-embedded tissue was sectioned and stained
with H&E and trichrome stain. The sections from
each specimen were examined blindly and independently by three authors, who selected the most
typical section from each specimen, and then examined
it to assess fibrocartilage formation, new bone formation, and tendon graft bonding to adjacent tissue. Each
of these characteristics was given a score according to
the scoring system (Table 1). The number of blood
vessels, including capillaries and muscularized vessels,
was counted microscopically, and quantitatively
assessed in six areas of tendon-bone junction from three
sections of each biopsy specimen. Each image was
captured by a digital camera (OLYMPUS C-5060;
Tokyo, Japan).
MATERIALS AND METHODS
Animal Surgery
Biomechanical Study
This study was approved by the Institutional Review
Board of Chang Gung Memorial Hospital, and performed under the guidelines for care and use of research
animals. Forty New Zealand rabbits weighing 3.2 1.6 kg were randomly divided into two groups. The HBO
group (n ¼ 20) was exposed to 100% oxygen at 2.5 atmospheres pressure for 2 h daily, for 5 consecutive days.
The control group (n ¼ 20) was housed in cages and
exposed to normal air. Twelve rabbits were used for
histological study, 24 rabbits for biomechanical
study, and four rabbits for analysis by electron microscopy (EM). Rabbits were anesthetized by an intravenous injection of 5 mL ketamine hydrochloride
(Ketalar; Parke Davis, Taiwan) and Rompum (Bayer,
Leverkusen, Germany) mixture. Medial parapatellar
arthrotomy was performed on the right hind limb
following a midline incision. The semitendinous tendon
was harvested from the ipsilateral limb with a tendon
stripper and the original ACL excised. Both femoral
and tibial tunnels were created with a drill bit at the
original insertion site. Reconstruction of the ACL with a
semitendinosus graft was performed by passing the
tendon graft into the knee joint through the bony
tunnels and securing graft to the adjacent periosteum
Twenty-four rabbits were sacrificed postoperatively:
12 rabbits each at 12 and 18 weeks. The knee joint,
DOI 10.1002/jor
Table 1. Scoring System for Histological Results
Characteristic
Points
Fibrocartilage formation
Abundant
Moderate
Slight
None
New bone formation
Abundant
Moderate
Slight
None
Tendon graft bonding to adjacent tissue
75 100%
0 75%
25 50%
0 25%
Perfect score
3
2
1
0
3
2
1
0
3
2
1
0
9
JOURNAL OF ORTHOPAEDIC RESEARCH MAY 2007
638
YEH ET AL.
including the femur, tibia, and tendon graft, was
harvested and remaining structures removed. Tensile
strength of the tendon graft was measured with a
Material Testing Machine (MTS system; Minneapolis,
MN). Femur and tibia bones were fixed with bone
clamps attached to the machine. Tendon graft tensile
strength was measured by a slow distraction (5 cm/s) of
the machine, and peak load was determined when graft
failure occurred. Sites and modes of graft failure,
including graft pullout from the bone tunnel, graft
rupture, and avulsion fracture at the tendon-bone
junction were assessed. The MTS tests were performed
on HBO and control group rabbits.
the intra-articular portion and from the intraosseous
tunnel were obtained from both groups. These specimens were fixed overnight in a 2% glutaraldehyde, 2%
paraformaldehyde, and 0.1 M sodium cacodylate buffer
solution. Specimens were then rinsed with 0.1 M sodium
cacodylate buffer followed by postfixation in 1%
osmium tetroxide in 0.1 M sodium cacodylate buffer.
After dehydration in graded alcohol, specimens were
embedded in epoxy resin. Finally, specimens were cut in
ultra-thin cross sections (60 nm) and stained with
uranyl acetate.
Statistical Analysis
EM Examination for Tendon Insertion
Four rabbits were sacrificed postoperatively at 18 weeks.
Specimens from the peripheral and central sections of
Tensile strength between the control and HBO groups
was compared using the Student’s t-test. Histological
results were analyzed using the Mann-Whitney U-test.
Values are expressed as means SD for Student’s t-test
Figure 1. Photomicrographs of 6-week specimens. A distinct tendon-bone interface
exists in both the control (A) and HBO (B) groups. Newly forming fibrocartilage lining the
bone tunnel is denser in the HBO group (B) than in the control group (A). At higher
magnification, the control group (C) shows less fiber-bony anchorage between the graft
and new fibrocartilage than in the HBO group (D). A higher number of Sharpey’s fibers
bridge the newly formed fibrocartilage and graft in the HBO group (F) than in the control
group (E). Trichrome stain: (A,B) ¼ 40; (C,D,E,F) ¼ 200.
JOURNAL OF ORTHOPAEDIC RESEARCH MAY 2007
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HBO TREATMENT ON TENDON GRAFTS IN RABBITS
639
Figure 1. (Continued )
and as medians for Mann-Whitney U-test; p < 0.05 was
considered statistically significant.
RESULTS
Surgery was successful in all studied animals and
no graft caused noticeable irritation or infection.
DOI 10.1002/jor
Histological Findings
Serial histological analysis of the tendon-bone
interface demonstrated that fibrovascular connective tissue, progressive collagen fiber-bone
anchorage, and fibrocartilage developed between
the graft and bone. At 6 weeks after surgery, grafts
were enclosed by fibrovascular connective tissue in
JOURNAL OF ORTHOPAEDIC RESEARCH MAY 2007
640
YEH ET AL.
both groups (Fig. 1A,B), but it was more organized
and mature in the HBO group (Fig. 1B). New
fibrocartilage formed around connective tissue in
both groups (Fig. 1C,D). Significant empty space
and less fiber-bony anchorage between the graft
and new fibrocartilage were noted in the control
group (Fig. 1C) compared to the HBO group
(Fig. 1D). Sharpey’s fibers were mainly present
in areas between the graft and the new fibrocartilage (Fig. 1E,F). A higher number of fibers
bridged the newly formed fibrocartilage and graft
in the HBO group (Fig. 1F) compared to the control
group (Fig. 1E). At 12 weeks (Fig. 2A,B), the
grafts in the HBO showed increased density and
organization with a considerable amount of fibrocartilage formed around the graft. Less fibrocartilage was found in the control group (Fig. 2C) but
more mineralized fibrocartilage was found in the
HBO group (Fig. 2D). At 18 weeks (Fig. 3A,B), new
bone had thickened and become more lamellar in
both groups. The interface fibrous layer was
integrated and mixed with tendon and bone lining,
which made it impossible to identify the margin.
Treatment by HBO significantly improved the
progressive interface incorporation of tendon graft
and bone (Fig. 3B).
Results for Histological Scoring System
Table 2 presents a summary of the histological
scoring system in the HBO and control groups.
The total scores were better for the HBO groups
than for the control groups (p < 0.05) at each time
point shown.
Figure 2. Photomicrographs of 12-week specimens. The HBO group (B) showed
increased density and organization of the tendon graft compared to the control group (A),
with a substantial amount of new fibrocartilage formation surrounding the graft. At
higher magnification, less fibrocartilage was found in the control group (C) than in the
HBO group (D), and more mineralized fibrocartilage was found in the HBO group (D).
Trichrome stain: (A,B) ¼ 40; (C,D) ¼ 200.
JOURNAL OF ORTHOPAEDIC RESEARCH MAY 2007
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HBO TREATMENT ON TENDON GRAFTS IN RABBITS
641
Figure 3. Photomicrographs of 18-week specimens. Mineralized fibrocartilage was
thicker and more lamellar in the HBO group (B) than in the control group (A). The interface
fibrous layer became integrated and mixed together with tendon and bone lining, making it
impossible to identify the margin in the HBO group (B). Trichrome stain: (A,B) ¼ 100.
Increase of Neo-Vessels after HBO Treatment
Figures 4 shows the microscopic features of neovessels of the HBO and control groups. Table 3
presents a summary of the numbers of neo-vessels
in the HBO and control groups. The number of
neo-vessels in the HBO group was significantly
DOI 10.1002/jor
increased postoperatively at 6 weeks (Fig. 4B) and
12 weeks (Fig. 4D).
Biomechanical Study
Table 4 presents tensile strength testing results at
the tendon-bone junction for the HBO and control
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642
YEH ET AL.
Table 2. Histological Score Results
Time
6 Weeks
12 Weeks
18 Weeks
Control
HBO Treatment
p Value
3,2,2
4,4,3
5,4,3
6,6,6
8,7,7
7,7,7
*p < 0.05
*p < 0.05
*p < 0.05
Mann-Whitney U-test.
groups. Similar modes of mechanical failure of the
tendon graft at the tendon-bone junction were
observed. Complete graft pullout from the bone
tunnel was noted in all cases in the control group,
where the modes of failure in the HBO group
included graft pullout in seven specimens and
graft breakage in five specimens at the tendonbone junction. The difference in the tensile
strength between the HBO and control groups
was statistically significant at 12 weeks (p < 0.01)
and 18 weeks (p < 0.01). Treatment by HBO
significantly increased the tensile strength at the
tendon-bone interface.
EM Findings
The fibroblast cells were oval to spindle shaped.
Cytoplasmic extension and development of intracellular organelles was moderate. The collagen
fibrils were in more uniform arrangement, were
more densely packed, and ran more parallel in the
HBO groups (Fig. 5B) than in the control groups
(Fig. 5A). At higher magnification, collagen fibrils
of the tendon graft were irregular and loose with
degraded and regenerated collagen fibers (Fig. 5C)
in the control group. Regenerated collagen fibers
after HBO treatment (Fig. 5D) were larger, more
Figure 4. Microscopic features of neo-vessels of the HBO groups (B,D) and the
control groups (A,C). A significantly increased number of neo-vessels was noted in
the HBO group at 6 weeks (B) and 12 weeks (D) after surgery. H&E stain:
(A,B,C,D) ¼ 100.
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HBO TREATMENT ON TENDON GRAFTS IN RABBITS
Table 3. The Effect of HBO Treatment on the
Ingrowth of Neo-Vessels
Time
Control
HBO Treatment
p-Value
1
3
3
10
*p < 0.05
*p < 0.01
6 Weeks
12 Weeks
Mann-Whitney U-test.
regular, and more compact than those in the
control group.
DISCUSSION
Successful ligament reconstruction depends, in
part, on the degree to which the tendon graft is
incorporated into bone. Numerous studies have
demonstrated that a tendon graft is incorporated
into a bone tunnel by ossification or formation of
a fibrous sleeve of callus, which thickens and
becomes more lamellar as the healing process
progresses.14–17 In clinical practice, most ligament
reconstructions of the knee are performed
with a free tendon graft such as patellar bonetendon-bone or hamstring graft. These grafts are
under physiological cyclic loading of the knee joint
during daily activities. The mechanism of incorporation of a graft to bone under physiological
loading condition is unknown.16,18–20 Previous
studies described the development of Sharpey-like
collagen fibers bridging the bone and the graft has
been described3,4,21 and is viewed as the earliest
sign of osseous integration.3,4,21 Rodeo et al.4 found
Table 4. The Effect of HBO Treatment on Tensile
Strength and Graft Failure
Time
Control
(n ¼ 6)
12 Weeks
Mean SD
46.8 6.3
Mode of failure
Pullout
6
Breakage
0
Fracture
0
18 Weeks
Mean SD
71.8 7.9
Mode of failure
Pullout
6
Breakage
0
Fracture
0
HBO Treatment
(n ¼ 6)
p-Value
69.6 8.9
*p < 0.01
4
2
0
96.4 10.2
*p < 0.01
3
3
0
Peak load (in Newtons) represents the tensile strength when
graft failure occurred.
Student’s t-test.
DOI 10.1002/jor
643
that the first Sharpey-like fibers developed at
4 weeks. Blickenstaff et al.5 and Grana et al.3
investigated semitendinosus tendon healing in an
intra-articular model in rabbits and found the first
Sharpey-like fibers at 3 weeks. In the present
study, Sharpey-like fibers were present in areas
between the graft and the new fibrocartilage. More
broad and organized fibers bridged the newly
formed fibrocartilage and graft in the HBO group
(Fig. 1F) than in the control group at 6 weeks
(Fig. 1E). A higher osseous activity was found in
the HBO group.
Remodeling processes of tendon grafts depend
upon numerous factors.16,20,22,23 Anderson et al.24
and Rodeo et al.20 demonstrated that adding
osteoinductive growth factors, such as bone
morphogenetic protein (BMP), improves tendon
healing in a bone tunnel. Other studies have also
shown that parathyroid hormone, HBO, and
physical factors such as continuous passive motion
(CPM) accelerate the healing of joint tissues,
including bone and cartilage.3,5,22,25,26
This study employed intra-articular tendon
transplantation mimicking ACL reconstruction
under physiological knee loading similar to that
in clinical applications. Experimental results
demonstrated that HBO treatment significantly
increased the amount of trabecular bone around
the tendon graft, increased the incorporation of
tendon and bone, and enhanced the graft tensile
strength. The exact mechanism of HBO on healing
remains unclear. Experimental results of our
previous animal studies showed that HBO treatment promotes27,28 and mitigates29 the adverse
effect of cigarette smoking29 on bone healing
following tibial lengthening in rabbits. Other
studies have also demonstrated that HBO accelerated the activity and rate of osteoinduction by
BMP,30,31 and induced angiopotein-2 expression
in endothelial cells.32 Furthermore, our present
experimental findings suggested that HBO
increased the angiogenesis in tendon graft and
tendon-bone interface (Fig. 4), and enhanced
tendon graft collagen fibril regeneration (Fig. 5),
therefore increasing the tensile loading strength of
the tendon graft. Therefore, it is reasonable to
assume that HBO treatment stimulates the
ingrowth of blood vessel formation associated with
improvement in blood supply that leads to an
increase in trabecular bone around the tendon
and improvement in the contacting between tendon
and bone at the tendon-bone interface. In conclusion, HBO treatment significantly improved the
healing of a tendon graft to bone in a bone tunnel in
rabbits.
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644
YEH ET AL.
Figure 5. Electron microscopy photomicrographs of 18-week specimens. The
fibroblast cells are oval to spindle shaped. Cytoplasmic extension and development
of intracellular organelles is moderate in the control groups (A) and the HBO
groups (B). At higher magnification, collagen fibrils of the tendon graft are irregular
and are loose with degraded and regenerated collagen fibers in the control group
(C). Collagen fibrils became larger, more regular, and were more compact with
predominantly new collagen fibers after HBO treatment (D). (A,B) ¼ 2,000;
(C,D) ¼ 4,000.
ACKNOWLEDGMENTS
This research was supported in part by grants from the
National Science Council and Chang Gung Memorial
Hospital, Taiwan, Republic of China.
JOURNAL OF ORTHOPAEDIC RESEARCH MAY 2007
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