Abstract
Background: Clinical studies have demonstrated a high rate of
incomplete healing of rotator cuff tendon repair. Since healing of such a
repair is dependent on bone ingrowth into the repaired tendon, we hypothesized
that osteoinductive growth factors would improve rotator cuff
tendon-healing.
Methods: Seventy-two skeletally mature sheep underwent detachment of
the infraspinatus tendon followed by immediate repair. The animals received
one of three treatments at the tendon-bone interface: (1) an osteoinductive
bone protein extract on a Type-I collagen sponge carrier, (2) the collagen
sponge carrier alone, and (3) no implant. The animals were killed at six and
twelve weeks, and the repaired rotator cuff was evaluated with use of magnetic
resonance imaging, plain radiographs, histologic analysis, and biomechanical
testing.
Results: A gap consistently formed between the end of the repaired
tendon and bone in this model, with reparative scar tissue and new bone
spanning the gap. Magnetic resonance imaging showed that the volume of newly
formed bone (p < 0.05) and soft tissue (p < 0.05) in the tendon-bone gap
were greater in the growth factor-treated animals compared with the collagen
sponge control group at both time-points. Histologic analysis showed a
fibrovascular tissue in the interface between tendon and bone, with a more
robust fibrocartilage zone between the bone and the tendon in the growth
factor-treated animals. The repairs that were treated with the osteoinductive
growth factors had significantly greater failure loads at six weeks and twelve
weeks (p < 0.05); however, when the data were normalized by tissue volume,
there were no differences between the groups, suggesting that the treatment
with growth factor results in the formation of poor-quality scar tissue rather
than true tissue regeneration. The repairs that were treated with the collagen
sponge carrier alone had significantly greater stiffness than the growth
factor-treated group at twelve weeks (p = 0.005).
Conclusions: This model tests the effects of growth factors on scar
tissue formation in a gap between tendon and bone. The administration of
osteoinductive growth factors resulted in greater formation of new bone,
fibrocartilage, and soft tissue, with a concomitant increase in tendon
attachment strength but less stiffness than repairs treated with the collagen
sponge carrier alone.
Clinical Relevance: This study is the first, as far as we know, to
demonstrate the possibility of increasing tissue formation in a tendon-bone
gap with use of a biologic agent. It shows the importance of the use of
magnetic resonance imaging to evaluate the repaired tendon, since the findings
on gross observation and even histologic examination could easily be
interpreted as representing an intact repair. Further studies with use of more
clinically relevant models of tendon-bone repair may support the use of growth
factors to improve clinical outcomes.
Injuries of the rotator cuff tendons in the shoulder are common in
individuals who engage in overhead activities for recreation or occupation.
These injuries frequently result in substantial pain and disability, and they
may require surgical repair. Repair of the rotator cuff typically involves
direct reattachment of the torn tendon to the bone of the proximal part of the
humerus. The attachment site between the rotator cuff tendon and the bone is
the so-called weak link, and because healing between the repaired tendon and
the bone is a slow process, prolonged periods of relative immobility are
required postoperatively. The resultant shoulder stiffness and weakness
typically require extensive physical therapy for restoration of full
function.
Despite careful postoperative management of rotator cuff tendon repairs,
objective evaluation of the repairs has demonstrated a high rate of incomplete
healing and gap formation between the tendon and the bone. Harryman et al.
used ultrasonography to evaluate rotator cuff repairs at an average of five
years postoperatively and found a recurrent cuff defect in 20% of isolated
repairs of the supraspinatus tendon and in >50% of the repairs that had
involved more than the
supraspinatus1. More
recently, Ball et al. used ultrasound to evaluate twenty arthroscopic repairs
of large chronic rotator cuff tears and found that 90% had a recurrent defect
despite good clinical
results2. In an
effort to improve the results of rotator cuff repair, Gerber et al. performed
a laboratory study to determine the repair technique with the greatest initial
fixation strength3.
The same authors later used magnetic resonance imaging to evaluate the repair
site, and reported a retear rate of 34% in twenty-nine patients at an average
of thirty-seven months
postoperatively4.
Importantly, the functional results following rotator cuff repair are
superior in shoulders in which the repaired cuff is intact at the time of
follow-up1,4.
Thus, it is imperative to develop methods to improve healing at the
tendon-to-bone junction. Numerous studies have focused on methods to improve
the initial fixation strength between the tendon and bone, with attention to
the type of fixation device (suture anchors, sutures alone, or newer fixation
devices), suture pattern, and type of arthroscopic
knot3,5,6.
However, there is very little information available about biological methods
to improve tendon-to-bone healing.
Experimental studies have demonstrated that healing between the repaired
rotator cuff tendon and bone is dependent on bone
ingrowth5,7.
Healing begins with the formation of a fibrovascular interface tissue between
the tendon and bone, followed by gradual bone ingrowth into this fibrous
interface tissue and then into the tendon, resulting in eventual
reestablishment of collagen fiber continuity between the tendon and bone.
There is a gradual increase in attachment strength as healing
progresses7.
Previous studies in our laboratory showed improved attachment strength of a
tendon graft in a bone tunnel in specimens in which there was greater bone
formation due to treatment with bone morphogenetic
protein8,9.
Because of the importance of bone ingrowth in tendon-to-bone healing and the
fact that rotator cuff tendon-healing is often limited by deficient tissue
formation with gap formation at the repair site, we hypothesized that
osteoinductive agents could improve the healing of a tendon attached to the
bone surface. We had three specific hypotheses: (1) osteoinductive agents
would induce greater formation of soft tissue, including fibrocartilage, at
the tendon-bone interface compared with untreated repairs; (2) osteoinductive
agents would induce greater bone ingrowth into the newly formed tissue in the
tendon-bone interface; and (3) improved soft-tissue and bone formation in the
treated specimens would result in a stronger tendon-to-bone attachment
compared with untreated repairs. We used a sheep model of rotator cuff tendon
repair to evaluate the effect of a combination of bone-derived growth factors
on the healing process.
Atotal of seventy-two skeletally mature female Rambouillet X Columbian
sheep were utilized for this study. This study was approved by the Colorado
State University Animal Care and Use Committee. Each animal underwent
unilateral detachment of the infraspinatus tendon followed by immediate
repair. The experimental group consisted of twenty-four animals that received
1.0 mg of an osteoinductive bone protein extract (Growth Factor Mixture
[GFM; Sulzer Biologics, Wheat Ridge, Colorado] described in detail
below) on a Type-I collagen sponge carrier applied to the tendon-bone
interface. There were two control groups: twenty-four animals that received
only the collagen sponge carrier with no growth factors and twenty-four
animals that underwent the tendon repair with no implant. A total of
thirty-six animals were killed at six weeks and thirty-six animals were killed
at twelve weeks (twelve in each of the three groups at each time). The
repaired rotator cuff was evaluated with use of plain radiographs, magnetic
resonance imaging, histologic analysis, and biomechanical testing. The dose of
the osteoinductive bone protein extract was chosen on the basis of a pilot
study performed by the study sponsor (Sulzer Biologics).
Surgical Procedure
The animal model and surgical technique that we used were identical to
those used in previously reported
studies5,10,11.
After the induction of general endotracheal anesthesia, the right shoulder was
prepared for sterile surgery. Anesthesia was induced with use of ketamine (4
mg/kg) and Valium (diazepam; 7.5 mg in total) and was maintained with
halothane (1.5% to 3.0%) in 100% oxygen (2 L/min). A transverse incision was
made over the lateral aspect of the right shoulder. The brachialis muscle was
split in line with its fibers to allow exposure of the underlying acromion.
The acromial head of the deltoid muscle was partially released from the
humerus, exposing the infraspinatus tendon. The infraspinatus tendon was
approximately 15 mm in width and very similar in size and thickness in each
animal. The tendon was sharply detached from the greater tuberosity. The
greater tuberosity was then prepared for repair of the tendon by removing any
remaining soft tissue and fibrocartilage. The greater tuberosity was lightly
decorticated with use of a high-speed burr until punctate bleeding from the
bone was noted. Decortication was done to a depth of only 1 mm; cortical bone
still remained. Four 2.0-mm drill-holes were placed into the greater
tuberosity for repair of the rotator cuff tendon to bone. These holes exited
laterally over the proximal humeral cortex. Two number-5 Ethibond sutures
(Ethicon, Somerville, New Jersey) were passed in a Mason-Allen configuration
through the tendon, and each suture was brought through one of the four
holes3,5.
The sutures were then tied over the lateral humeral cortex over a
stainless-steel cortical bone augmentation plate (Synthes, Paoli,
Pennsylvania)3. This
allowed a secure repair of the tendon to the greater tuberosity. Routine wound
closure was then performed. At the time of surgery, the collagen sponge
implant either with or without GFM was applied to the tendon-bone
interface prior to securing the sutures
(Fig. 1). In the second control
group, no implant was placed at the tendon-bone interface.
All surgery was performed on the right shoulder only. Perioperative
antibiotics (1 g of cefazolin) were administered.
Prior to awakening the animal from anesthesia, a large softball was taped
to the hoof on the operative side in order to discourage weight-bearing on the
limb. This method of postoperative immobilization has been used by other
investigators5,10.
The ball was kept on the hoof for five weeks, although it was evident that the
animals did bear some weight on the involved limb. Postoperative analgesia was
provided by phenylbutazone (1 g given orally) for three days following
surgery, and a fentanyl patch (Duragesic [50 µg/h]; Janssen
Pharmaceuticals, Titusville, New Jersey) was worn. The animals were returned
to their pens immediately following surgery and were generally able to walk
within twenty-four to forty-eight hours. A standardized scale from 0 to 17,
which was based on animal alertness, movement, flock behavior, feeding
behavior, and respiratory rate, was used to assess postoperative pain.
Experimental Implant
The osteoinductive bone protein extract (Growth Factor Mixture
[GFM]) was produced by Sulzer Biologics. This material was obtained
from bovine cortical bone as described
previously12.
Briefly, the process involved removal of all soft tissue and marrow from the
bone, after which the bone was pulverized and then demineralized in 1 N of
hydrochloric acid for eight hours at 25°C. The demineralized bone was
washed in deionized water and extracted with a 4-M guanidine hydrochloric acid
solution buffered with 0.01-M Tris at pH 7.6 for forty-eight hours at
15°C. The extracted proteins were purified with a 100,000-molecular-weight
cutoff ultrafilter, followed by a 10,000-molecular-weight cutoff ultrafilter.
Final purification was done with use of ion-exchange chromatography and
reverse-phase high-pressure liquid chromatography. Electrophoretic migration
patterns of this extract demonstrate bands in the range characteristic of bone
morphogenetic proteins 2 through 7 (BMP-2-7), transforming growth
factor-ß-1-3, and fibroblast growth factor. Subcutaneous implants of this
material induced bone formation in
rats12.
Osteoinductive bone protein (1.0 mg) was added to a Type-I collagen sponge
carrier (20 mm × 10 mm × 1.7 mm) and then lyophilized. The
collagen sponge delivery vehicle was Type-I collagen derived from bovine
tendon (ReGen, Franklin Lakes, New Jersey).
Thirty-six animals were killed at six weeks, and thirty-six animals were
killed at twelve weeks. Euthanasia was performed according to the guidelines
set forth by the American Veterinary Medicine Association Panel on
Euthanasia13. At
each time-point there were twelve animals in each group. At the time that the
animals were killed, anteroposterior and lateral radiographs were made of each
limb and then the tissue was frozen and shipped to our institution for further
evaluation. Of each group of twelve shoulders, six were randomly chosen for
magnetic resonance imaging. After magnetic resonance imaging, three shoulders
were prepared for histological analysis and nine were prepared for
biomechanical testing.
Magnetic Resonance Imaging
We tested our hypotheses about the volume of healing tissue and new bone
ingrowth using magnetic resonance imaging and histological analysis. Magnetic
resonance imaging was carried out for six animals in each group at each
time-point. Images were performed in a clinical 1.5-T superconducting magnet
(Signa Horizon LX; General Electric Medical Systems, Milwaukee, Wisconsin)
with use of a commercially available, receive-only shoulder phased-array coil
(shoulder array; Med Rad, Indianola, Pennsylvania). Oblique axial images were
acquired through the long axis of the rotator cuff tendon with use of a
fast-spin-echo pulse sequence, with a repetition time of 4000 to 5500 msec, an
effective-echo time of 29.8 to 34.7 msec, and a receiver bandwidth of 31.2 to
62.5 kHz, at three excitations. The field of view ranged between 13 and 14
cm2 to allow for visualization of the muscle-tendon junction and
tendon-to-bone attachment, and the matrix was 512 × 352, yielding an
in-plane resolution of 254 µ in the frequency direction by 369 µ in the
phase direction; slice thickness was 1.6 mm with no interslice gap. Pulse
sequence parameters were chosen for sensitivity to fluid, to serve as an
internal comparison for signal properties of the tendon. On all pulse
sequences obtained, fluid in the glenohumeral joint had high signal intensity.
The wider receiver bandwidth was chosen to minimize the frequency shift
generated by the susceptibility artifact from the nonabsorbable sutures and
surrounding plate. No frequency-selective fat suppression was utilized.
The magnetic resonance images were evaluated by a radiologist blinded to
the presence or absence of the experimental implant. In order to determine
precisely the complete volume of tissue in the tendon-bone gap, magnetic
resonance image files were transferred onto a standard personal computer, and
a manual segmentation of the interface was performed. Subsequent conversion of
pixels to cubic millimeters was performed with use of conversion software and
a program developed on Mat Lab 6.2 software (Applied Science Laboratory;
General Electric Medical Systems). The program calculates the volume of tissue
in the tendon-bone gap by the summation of the areas normally demarcated on
each slice, with use of the formula: volume = S (Ar × ST), where
Ar is the area of tissue on the slice and ST is the slice thickness. The
tendon-bone gap tissue was defined as the tissue between the end of the tendon
and the bone; this tissue was easily distinguished from the bone and the
native tendon. We derived an estimate of the tissue material properties by
dividing the biomechanical measurements (ultimate load and stiffness,
described below) by the measurement of tissue volume in the tendon-bone
gap.
Other magnetic resonance imaging parameters that were graded included (1)
the signal characteristics of the cancellous bone at the humeral head repair
site (normal or hyperintense); (2) the signal characteristics of the
tendon-bone gap tissue, i.e., low signal intensity (isosignal intense to
"normal" tendon), intermediate signal intensity (isosignal intense
to skeletal muscle), and high signal intensity (isosignal intense to fluid in
the glenohumeral joint); (3) the thickness of the end of the tendon
(anteroposterior dimension); and (4) the width of the gap measured from the
greater tuberosity repair site to the lateral edge of the tendon. Gap
measurements were made on five or six serial images and then were averaged.
Relative signal intensity measurements were performed on an Advantage Windows
workstation (General Electric Medical Systems), with use of a relative scale
of signal intensity acquired directly from the magnetic resonance images. A
standardized 1-mm2 region of interest was sampled from the tendon
remote from the site of repair, as well as from three 1-mm2 samples
from the tissue in the tendon-bone gap. These three values were then averaged.
The signal intensity was measured relative to other tissues in the joint on
the same image, thus providing an internal control.
Histological Analysis
The tissues for histological analysis were fixed in 10% neutral buffered
formalin for approximately two weeks and then were decalcified in 5% nitric
acid. After the tissue was fully decalcified, the tissue was further trimmed
and embedded in paraffin. Five-micrometer-thick sections were made and were
stained with hematoxylin and eosin and safranin O. Coronal sections were made
in line with the tendon, which allowed evaluation of the bone, the tendon-bone
gap tissue, and the end of the repaired tendon. The histological sections were
viewed with use of light and polarized light microscopy on an Olympus BH-2
microscope (Olympus Optical, Lake Success, New York). The sections were
evaluated in a blinded fashion with no knowledge of experimental or control
group. We assessed new-bone formation at the greater tuberosity, cellularity
and vascularity in the tendon-bone gap tissue, new matrix deposition in the
tendon-bone gap, the presence of cartilage in the tendon-bone gap, and
collagen fiber continuity and so-called organization from the bone surface
into the gap tissue.
Biomechanical Testing
We tested our third hypothesis about the tendon attachment strength, using
standard biomechanical testing protocols. At the time of biomechanical
testing, the specimens were thoroughly thawed. All muscle was removed from the
infraspinatus, but the tendon itself was left intact. A specially designed jig
was made for mounting the proximal part of the humerus. Testing was carried
out on a load frame (MTS, Eden Prairie, Minnesota) coupled to a controller
(Instron, Canton, Massachusetts). The specimens were mounted in the load frame
for uniaxial tensile loading in line with the pull of the infraspinatus
tendon. The proximal part of the humerus was mounted and secured with clamps,
and then the tendon was gripped in a specially fabricated gripping device with
serrated edges that allowed secure gripping of the soft tissue and prevented
slippage. The tendon was gripped at a constant distance (25 mm) from the bone
in each specimen. Data were collected on a personal computer with use of data
acquisition software (Lab-Tech Notebook, Andover, Massachusetts). The
load-frame data were collected at a rate of 20 Hz. All testing was carried out
at room temperature, and the specimens were kept moist with normal saline
solution during testing.
Each specimen was cycled between 10 N and 60 N (approximately 1% strain) at
a rate of 1 Hz for a total of ten cycles to precondition the specimens prior
to loading. Each specimen was then loaded to failure at a cross-head
displacement rate of 20 mm per minute. The ultimate load-to-failure and the
site of failure were recorded for each specimen. Grip-to-grip displacements
were used to compute stiffness.
Data Analysis
Our primary purpose was to compare the growth factor-treated group with the
control groups. As a secondary analysis, we made comparisons within each group
over time. The volume of new bone and new soft-tissue formation at the
tendon-bone attachment site and the signal intensity measurements in the
tendon and the interface (as measured by magnetic resonance imaging) were
compared with use of Kruskal-Wallis rank analysis of variance (version 11.0;
SPSS for Windows, Chicago, Illinois). For the biomechanical data, the ultimate
failure load and stiffness in the control and experimental groups were
compared at each time-point with use of analysis of variance for normally
distributed data and a Kruskal-Wallis rank analysis of variance for
non-normally distributed data. Post hoc tests were done with use of the
Mann-Whitney rank-sum test and Bonferroni corrected comparisons. The same
statistical tests were used to compare the six-week and twelve-week data
within each group. Correlations between the biomechanical data and the
magnetic resonance imaging measurements were performed with use of linear
regression analysis. Significance was set at p < 0.05.
Gross Observations
All animals tolerated the surgical procedure well with no intraoperative or
perioperative complications. There was no evidence of infection or
immunological reaction in any specimen. By postoperative day 2, the pain
scores were 0 to 1, and by postoperative day 3, the scores were 0 in all
animals.
Direct observation of the specimens showed reparative scar tissue spanning
a gap between tendon and bone in all specimens. It was difficult to discern
scar tissue from normal tendon by gross observation. Gross observation
demonstrated a greater volume of new tissue formation at the tendon attachment
site in the growth factor-treated animals, and some specimens had extensive
new-bone formation at the tendon repair site at the greater tuberosity.
Comparison of Experimental and Control Animals
Radiographs
Plain radiographs demonstrated new-bone formation at the greater tuberosity
as well as over the cortical bone augmentation plate in the growth
factor-treated specimens. This new bone appeared to be remodeled over time,
such that less new bone was present at twelve weeks compared with six weeks.
New bone was not present in either of the control groups. There was no
evidence of substantial bone resorption at the tendon repair site on plain
radiographs.
Magnetic Resonance Imaging
Magnetic resonance imaging allowed clear distinction between the end of the
repaired tendon, the newly formed tissue in the tendon-bone gap, and the
greater tuberosity (Figs. 2-A,
2-B, and
2-C). The tendon had normal
low-signal intensity, while the newly formed tissue between the tendon and
bone had intermediate to high-signal intensity. There was extensive periosteal
new-bone formation and increased signal intensity in the humeral head in the
growth factor-treated group compared with controls. In the six-week group, the
average gap (and standard deviation) between the repaired tendon and bone was
23.0 ± 5.6 mm in the controls with no implant, 25.9 ± 5.6 mm in
the collagen-carrier control, and 19.3 ± 4.6 mm in the growth
factor-treated group; the differences were not significant. In the twelve-week
group, the average gap between the repaired tendon and bone was 22.9 ±
10.4 mm in the controls with no implant, 24.0 ± 12.1 mm in the collagen
carrier control, and 19.1 ± 11.9 mm in the growth factor-treated group;
the differences were not significant.
Magnetic resonance imaging demonstrated a significantly greater volume of
new-bone formation in the tendon-bone gap in the growth factor-treated group
at both six weeks and twelve weeks compared with the collagen carrier control
group (p < 0.05). Similarly, there was a significantly greater volume of
newly formed soft-tissue in the tendon-bone gap in the growth factor-treated
group compared with the collagen carrier control at both six weeks (p <
0.05) and twelve weeks (p < 0.05) (Fig.
3).
There was significantly greater magnetic resonance imaging signal intensity
in the interface tissue in the collagen carrier control at six weeks (p <
0.05), but this was no longer significant at twelve weeks. There were no
significant differences in signal intensity in the tendon itself between the
controls and the growth factor-treated specimens at either six or twelve
weeks.
Histological Analysis of the Repaired Tendon
Six-Week Specimens
The normal infraspinatus tendon in the sheep inserts to the bone by means
of a direct insertion, with a zone of fibrocartilage between the tendon and
bone. In this animal model, the gap between the tendon and bone fills in with
fibrovascular granulation tissue in both the controls and growth
factor-treated specimens. A completely normal-appearing insertion site was not
reformed in any group. The infraspinatus tendon itself appeared essentially
normal, with viable cells and well-organized collagen fibrils in both groups.
The native tendon could be easily distinguished from this interface scar
tissue. In the control specimens, the newly formed tissue in the tendon-bone
interface gap was highly cellular, containing a mix of spindle-shaped
fibroblastic cells, mononuclear cells, and occasional chondrocytes (Figs.
4-A,
4-B,
4-C, and
4-D). The collagen fibers in
the interface tissue between the tendon and bone were moderately well
organized. At the bone surface, there was a thin seam of newly formed woven
bone, and in some specimens there were small areas of cartilage in the
interface. In contrast, the growth factor-treated specimens demonstrated
extensive new-bone and cartilage formation in the tendon-bone gap by six
weeks. There was a greater volume of interface tissue in the growth
factor-treated specimens than in both of the control groups. This tissue was
heterogeneous, with some areas moderately well organized (collagen fibers
aligned in the same direction) and other areas poorly organized. No remnants
of the collagen sponge could be identified histologically in either sponge
group at six weeks.
Twelve-Week Specimens
By twelve weeks, the interface tissue in both controls and growth
factor-treated specimens was denser because of an increased matrix deposition
and was less vascular and less cellular than at six weeks. The collagenous
matrix was more oriented in line with the tendon in all three groups. In both
control groups, there were occasional small areas of cartilage in the
tendon-bone gap, but the gap was mostly filled with fibrous tissue (Figs.
5-A and
5-B). Polarized light
microscopy showed collagen fiber continuity between the tendon and bone.
Although some specimens in both control groups had a small zone of
fibrocartilage between the tendon and bone, a normal-appearing direct tendon
insertion site did not consistently reform by twelve weeks. The mineralized
fibrocartilage zone and the tidemark (mineralization front) that are found in
the normal direct tendon insertion site were not reformed in the specimens in
either control group.
In the growth factor-treated specimens at twelve weeks, the gap tissue
demonstrated well-organized collagen fibers that were oriented in line with
the tensile pull of the tendon and this tissue was less cellular and more
organized than that in the six-week specimens. The large areas of new bone and
cartilage that were present at six weeks had been remodeled by twelve weeks
and were more organized. The cells in the gap tissue also became aligned with
the tensile load on the tendon. Polarized light microscopy demonstrated
collagen fiber continuity from the bone surface into the fibrocartilage gap
tissue. The cartilage in the tendon-bone gap had remodeled into a more
normal-appearing fibrocartilage zone (Figs.
5-C and
5-D). There was more cartilage
formation in the tendon-bone gap in the growth factor-treated specimens
compared with both control groups. However, the columnar arrangement of
chondrocytes in a normal direct insertion was not consistently reestablished,
and the resulting insertion site still did not consistently demonstrate the
histological criteria of a normal direct insertion. The collagen sponge had
completely resorbed by twelve weeks in all animals.
Biomechanical Testing
All of the specimens failed at the soft tissue-to-bone attachment site. In
several specimens, small bone spicules were found on the end of the soft
tissue after failure. The ultimate load-to-failure was significantly higher in
the growth factor-treated group compared with the collagen carrier control at
six weeks (p < 0.05) and was significantly higher in the growth
factor-treated group compared with both control groups at twelve weeks (p <
0.05). When the failure load data were normalized by dividing by the volume of
new tissue at the attachment site, no significant difference was found among
the groups at either six weeks or twelve weeks (Figs.
6-A and
6-B).
The collagen control group was significantly stiffer than the growth factor
group (p = 0.005) at twelve weeks but did not differ from the controls with no
implant (p = 0.07); no significant difference was found between the growth
factor group and the controls with no implant
(Fig. 7). At the six-week
time-point, there was no significant difference among the groups.
We found a significant correlation between the normalized load to failure
and the volume of new soft-tissue in the tendon-bone gap at both six weeks
(r2 = 0.65, p = 0.009) and twelve weeks (r2 = 0.37, p =
0.02). There was a trend for a correlation between load to failure and the
tendon-bone gap distance (r2 = 0.42, p = 0.06).
Changes Over Time within Each Group
There was gradual maturation of the healing tissue from six weeks to twelve
weeks in all three groups, with more marked changes occurring in the growth
factor-treated specimens. This reflected the fact that there was extensive
formation of immature reparative scar tissue in the growth factor-treated
specimens at the early time-point. As described above, the scar tissue in the
tendon-bone gap became less cellular, with increased matrix deposition and
improved organization. There was a decreased volume of new-bone formation in
the tendon-bone gap in the growth factor-treated group at twelve weeks
compared with six weeks, while there was no significant change in the volume
of new soft-tissue formation in the tendon-bone gap between six weeks and
twelve weeks as measured by magnetic resonance imaging
(Fig. 3). There was no
significant difference in the signal intensity in the gap tissue or native
tendon between six and twelve weeks in any group.
There were significant increases in ultimate load-to-failure between six
weeks and twelve weeks in the growth factor-treated group (p < 0.05) and
the controls with no implant (p < 0.05), but there was no change in the
collagen carrier control group (Figs.
6-A and
6-B). Within the collagen
carrier control group, the twelve-week specimens were significantly stiffer
than the six-week group (p = 0.04); no differences were found between the
six-week and twelve-week time-points within either the growth factor-treated
group or the controls with no implant
(Fig. 7).
Failure of secure healing between tendon and bone is a major problem in
rotator cuff repair. Various factors are thought to account for poor healing
of the rotator cuff tendon, including intrinsic tendon degeneration, fatty
infiltration of the muscle and tendon, muscle atrophy, poor bone quality, and
weak tendon-to-bone fixation. Our goal in this study was to examine the
hypothesis that osteoinductive growth factors would improve healing of the
rotator cuff tendon to bone. Despite the use of the repair technique that has
been found to have the greatest biomechanical strength in in vitro models, the
repaired tendon consistently detached from the repair site in this animal
model3,4.
Thus, in this study, we modeled tissue formation in a gap between tendon and
bone. The gap was evident only by magnetic resonance imaging. Notably, gross
inspection of the specimens demonstrated stout tissue connecting the
infraspinatus muscle to the bone, with no evidence of a gap. Furthermore,
histological analysis showed a well-organized, tendon-like fibrous tissue
connecting to bone; the histological findings could easily be interpreted as
representing an intact repair. If magnetic resonance imaging were not
performed, it may have been concluded that the tendon remains firmly attached
to the bone. We believe that this is an important finding, as other
investigators are using this same model to evaluate healing of the rotator
cuff
tendon5,10,11,14.
On the basis of our results, we urge caution in interpreting the results of
other studies that have used a sheep model for rotator cuff repair.
It is likely that the tendon detached from the repair site in the early
postoperative period because of an inability to control the loads on the
repaired tendon immediately postoperatively. The resulting gap subsequently
filled in with a fibrovascular scar tissue. This scar tissue gradually matured
and acquired substantial load-bearing capacity. Other investigators have also
reported gap formation at a tendon-to-bone repair site. Gerber et al. used the
same animal model with an identical suture pattern and a cortical bone
augmentation plate, and they also found that the gap between the end of the
repaired tendon and the bone filled in with scar tissue that was often
indistinguishable from normal tendon. They termed this a "failure in
continuity."5
Similar to our results, they reported that the scar tissue in the defect was
composed of well-organized, dense collagen bundles by six months and was
difficult to distinguish from the end of the tendon.
Although we were not able to study healing of an intact rotator cuff tendon
repair, the resultant model of healing in a tendon-bone gap has clinical
relevance. Clinical studies of rotator cuff repair have demonstrated a high
rate of incomplete healing and gap formation between the tendon and the
bone1,2,4.
There are important concerns among shoulder surgeons about the strength of
rotator cuff tendon-to-bone fixation and the potential for formation of a
tendon-bone gap and failure of healing. Furthermore, various materials have
been developed to use as a scaffold to bridge a gap in rotator cuff tendon
repair15,16.
The ability to improve tissue formation in a tendon-bone gap may allow
improved clinical results.
Our results support our hypotheses about the induction of new soft-tissue
and bone formation. To our knowledge, this is the first report of the use of
growth factors to augment tissue formation between the rotator cuff tendon and
bone. Examination of the group with no implant showed that healing occurs in
this animal model by formation of a fibrovascular scar-tissue interface
between tendon and bone. Although there was partial reformation of a
fibrocartilage interface in the growth factor-treated animals, we did not
observe consistent formation of a normal insertion site. The growth factors
appeared to induce a greater reactive scartissue response rather than
regeneration of a morphologically normal insertion site. Although there was
abundant new tissue formation, the quality of this tissue was relatively poor,
as evidenced by the finding that at twelve weeks the failure loads were only
approximately 31% of the normal strength of a sheep infraspinatus tendon
insertion, on the basis of previously reported
data11. Magnetic
resonance imaging demonstrated persistently high signal in the reparative
tissue in the tendon-bone interface, reflecting increased mobile water and
indicative of a relatively poorly organized matrix. The growth factor-treated
group had significantly greater failure loads compared with both control
groups; however, when the data were normalized by tissue volume to provide an
estimation of material properties, there were no differences between groups.
Furthermore, the stiffness of the tendon-bone construct was not improved in
the growth factor-treated group compared with the controls. These results all
support the conclusion that the growth factor treatment results in the
formation of poor-quality scar tissue rather than true tissue regeneration.
Previous studies of the effect of growth factors on tendon and
ligament-healing have also described the formation of new tissue with inferior
material
properties17,18.
The reparative tissue in our model underwent remodeling and became more mature
over time, as demonstrated histologically by improved matrix organization and
biomechanically by increased attachment strength in the growth factor-treated
group between six and twelve weeks.
There were important differences between the groups that received the
collagen sponge implant (the collagen carrier control group and the growth
factor group). The stiffness was higher in the collagen carrier control group
compared with the growth factor group at twelve weeks. At the same time, we
found a lower volume of new tissue formation in animals receiving the collagen
sponge alone. The collagen sponge appeared to inhibit excessive tissue
formation. The presence of the sponge directly between tendon and bone may
have acted as a physical block to tissue formation. We also found increased
magnetic resonance imaging signal intensity in the tendon-bone interface in
the collagen sponge group at six weeks. It is possible that a subtle immune
response against the bovine-derived collagen sponge contributed to the
apparent inhibition of tissue formation and the increased magnetic resonance
imaging signal intensity, although histological evaluation did not support
that. The diminished new tissue formation in the collagen sponge group makes
the finding of new tissue formation in the growth factor-treated animals even
more notable.
The differences between the collagen carrier control group and the growth
factor group suggest distinctly different biologic responses to these
implants. We hypothesize that the collagen sponge by itself acts as a scaffold
to orient newly forming fibrous tissue. Because a gap forms in this model, the
sponge by itself may play a positive role in healing (as evidenced by improved
stiffness). The collagen sponge with the growth factor induces a vigorous
biologic response, leading to excessive new tissue formation. However, this
newly forming tissue has poor material properties at twelve weeks. We further
hypothesize that any beneficial effect on healing due to the collagen sponge
alone may not have been evident in the group that received a collagen sponge
with growth factor because of more rapid sponge resorption in this group. The
intense biologic response to the growth factors (including an early
inflammatory response) would likely accelerate sponge resorption. Analysis of
earlier time-points would be required to examine the kinetics of sponge
resorption. These findings have clinical relevance since there are currently
available collagen-containing extracellular matrix materials that are used as
a scaffold to improve rotator cuff tendon-healing. Similar to the findings in
our study, Schlegel et al. recently reported improved stiffness but no change
in failure loads with use of a swine small intestine submucosa patch to
augment rotator cuff tendon-healing in the same sheep
model19.
The potential for osteoinductive growth factors to improve rotator cuff
tendon repair is supported by previous studies that have examined healing of
tendon to bone. Although the basic cellular and molecular mechanism of rotator
cuff tendon-to-bone healing is poorly understood, previous animal studies have
shown that healing proceeds by bone ingrowth into the interface between tendon
and bone. Gerber et al. used a sheep model of infraspinatus tendon repair and
reported gross and histological findings very similar to our results, with an
osteoblastic response at the tendon-to-bone attachment
site5. Uhthoff et
al. used a rabbit model of supraspinatus tendon repair and found that, while
there was little cellular proliferation in the tendinous stump, there was
cellular and vascular proliferation within the underlying bone and subacromial
bursa at two weeks following
repair20. Further
support for the use of osteoinductive growth factors is provided by studies
that have demonstrated bone resorption and potentially impaired bone formation
at tendon and ligament insertion sites. For example, Kannus et al. reported
regional osteoporosis in the proximal part of the humerus in patients with a
rotator cuff tendon
tear21. Regional
osteoporosis in the proximal aspect of the humerus not only diminishes the
pull-out strength of sutures or bone anchors but also may result in impaired
bone formation at the healing tendon-bone
junction22. The use
of an osteoinductive agent at the insertion site may ameliorate these
deleterious bone changes.
There are limitations to the animal model used in this study. As discussed
above, the repaired tendon consistently detached from the repair site. In
humans, when a rotator cuff tendon repair fails, there is a persistent gap in
the tendon, whereas in the sheep model the repair site is extrasynovial and
the gap fills with scar tissue. It appears that the sheep is able to produce
abundant new bone at the greater tuberosity, which is unlikely to occur in the
typical patient undergoing rotator cuff repair who may be elderly and have
poorer bone-forming potential. Another limitation of this model is that in
this study we examined healing of an acute rotator cuff repair, which does not
mimic the typical clinical situation in which there is retraction and atrophy
of the torn tendon. There are typically intrinsic degenerative changes in the
torn rotator cuff tendon (tendinosis), tendon retraction, and osteoporosis of
the greater tuberosity in shoulders that have a long-standing rotator cuff
tear. The healing capacity of the tissue in these clinical situations is
likely diminished. Gerber et al. performed delayed repair of a retracted
tendon in the sheep model and reported an exceedingly high failure rate after
repair, which they attributed to the high tension on the
repairs5. Soslowsky
et al. developed a rat model of overuse tendinosis, which may be useful to
evaluate the effect of growth factors on rotator cuff
tendon-healing23.
Although the rat model has been used to study tendon repair, it is not known
whether repair failure and scar formation also occur in the rat.
Our results should be considered preliminary and only a first step toward
the development of growth factors for clinical application in rotator cuff
repair. We had a relatively small sample size, resulting in low power for some
comparisons. These findings would currently have limited application to
rotator cuff repair in patients, as a hypertrophic tissue response in the
subacromial space could lead to subacromial impingement. The abundant new
tissue likely forms in this model to fill the tendon-bone gap. It is possible
that such a hypertrophic tissue response may not occur if a gap does not form.
Further animal model development is necessary to test the effect of growth
factors on tendon-to-bone healing.
In conclusion, we found that a mixture of osteoinductive growth factors
leads to increased formation of new bone and soft tissue in a tendon-bone gap,
resulting in a stronger attachment between the tendon and bone at six and
twelve weeks after repair. We also found improved stiffness of the repairs
treated with a collagen scaffold alone. Because clinical studies of rotator
cuff repair have demonstrated a relatively high prevalence of failure of
complete healing of rotator cuff repairs, the use of extracellular matrix
scaffolds and growth factors to augment healing may provide a clinically
important improvement in rotator cuff repair. The use of biologic agents to
improve healing is likely to be especially valuable in patients with
diminished biologic healing potential due to rotator cuff tendinosis and
associated osteoporosis of the greater tuberosity. ?
Note: The authors thank Ian Tsou, MD, and Li Foong Foo, MD, for
their assistance with the interpretation of the magnetic resonance images, and
Stephen Lyman, PhD, for his assistance with the statistical analyses. The
authors also thank Deirdre Campbell, MEng, Kate Myers, BS, and Xiang-hua Deng,
MD, for technical assistance with biomechanical testing, and Jason
Schneidkraut, MD, for assistance with final manuscript preparation.
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