Overview of Study Design
One hundred twenty-six rats were randomly divided into six equal groups of
twenty-one animals each. Each rat was subjected to surgical excision of a
1.0-cm2 section of the musculotendinous portion of the ventral
lateral abdominal wall, with the abdominal fascia, transversus abdominis, and
peritoneum being left intact. The defect was then repaired with one of five
extracellular matrix-derived scaffold materials or autologous tissue,
depending on the assigned group. The animals in each group were subdivided
into seven groups based on survival time: two, four, seven, fourteen,
twenty-eight, fifty-six, or 112 days. At the time when the animal was killed,
the surgical site along with an equal amount of surrounding tissue was
collected for histological examination.
Test Articles
The five extracellular matrix-derived scaffold materials that were used to
repair the surgically created defect were all derived from biologic sources
and differed with regard to species of origin, tissue of origin, processing
methods, and/or method of terminal sterilization
(Table I and Appendix). Freshly
harvested autologous excised abdominal wall tissue served as the control
device. The commercially available devices are processed by methods that
disrupt cell membranes and cytoplasmic organelles, denature cytoplasmic
proteins, and largely eliminate these components from the remaining
extracellular matrix.
Each of the test articles evaluated in the present study (excluding
autologous abdominal wall tissue) was purchased from the manufacturer. The
handling of the materials after removal from the package and prior to surgical
implantation was performed according to the manufacturer's instructions as
provided in the package insert.
Animal Model
An established rat model of abdominal wall repair was used to evaluate the
host morphologic response to each implanted
biomaterial33. This
model includes muscle and tendon tissue (i.e., the musculotendinous junction),
the presence of a variable and multidirectional load, and a clinically obvious
failure outcome (i.e., hernia). All procedures described in the present study
were performed in accordance with the National Institutes of Health guidelines
for care and use of laboratory animals. All procedures were approved by the
Institutional Animal Care and Use Committee at the University of
Pittsburgh.
Sprague-Dawley rats weighing 300-500 g were purchased from Charles River
Laboratories (Wilmington, Massachusetts). The animals were housed individually
in shoebox cages and were fed a diet of Purina Isopro ad libitum. The bedding
was changed at least once per week. The animals were examined by a
veterinarian before surgery and were determined to be in good health. The
housing environment was maintained at a temperature of 68°F to
76°F.
Each animal was anesthetized with isoflurane (2% in oxygen) in an
inhalation chamber. The surgical site was clipped, shaved, and prepared for
sterile surgery with a Betadine (povidone-iodine) scrub. Sterile technique was
used at all times. A ventral midline abdominal incision was made, and the skin
and subcutaneous tissue were separated from the underlying muscle tissues on
one side of the midline for a distance of approximately 4.0 cm. The incision
in the ventral midline of the abdominal skin was retracted to expose the
ventral lateral wall adjacent to the linea alba, including the
musculotendinous junction of the abdominal wall musculature.
A 1.0-cm2 defect of the musculotendinous portion of the ventral
lateral abdominal wall was excised, with the underlying transversalis fascia
and peritoneum being left intact. Uniformity of the defect size and shape was
ensured by using a device with a fixed size and shape on each animal. The
defect was then replaced with a 1-cm2 piece of the test article
chosen for that animal. Autologous excised abdominal wall tissue served as the
control implant. One 4-0 Prolene suture was placed at each of the four corners
of the test article to secure attachment to the adjacent abdominal wall and to
demarcate the implant. Securing the test articles in this manner provided a
mechanism by which the test article was subjected to the mechanical forces
delivered by the adjacent native abdominal wall musculature, while avoiding
the predominance of a host-tissue reaction to the suture material rather than
the test article. There was no difficulty in manipulating or handling any of
the test articles in the study. A subcuticular placement of 4-0 Vicryl was
used to close the skin incision. Each animal recovered from anesthesia on a
heating pad and was returned to its housing unit.
Each rat received 0.02 mg Buprenex (buprenorphine hydrochloride) and 2 mg
of gentamicin subcutaneously immediately after surgery and for two and three
days afterward, respectively. The surgical site was evaluated daily for the
duration of the study, and any signs of swelling, discoloration, or herniation
at the operative site were recorded. The dietary habits and general health
status of each animal were recorded daily.
On the date on which it was scheduled to be killed, each rat was
anesthetized with isoflurane (5% in oxygen) followed by an intracardiac
injection of 5 mL of potassium chloride to induce immediate cardiac
arrest.
Immediately after the animal was killed, the defect site along with an
equal amount of adjacent native tissue was excised, mounted on a fixed support
structure, and placed in 10% neutral buffered formalin. The specimen was then
sectioned through its entire thickness and length, including generous amounts
of the adjacent normal body wall. The tissue was embedded in paraffin, cut
into 6-µm thick sections, and mounted on glass slides. The tissue was
stained with either hematoxylin and eosin or Masson trichrome before
coverslipping.
Semiquantitative histopathologic analysis included evaluation of (1) the
amount of cellular infiltration, (2) the presence or absence of multinucleated
giant cells, (3) vascularity, and (4) the degree of organization of the
replacement connective tissue. At forty times magnification, the cellular
infiltration counts were based on ten preselected sites at the host-device
interface (Table II).
Multinucleated giant cells, vascularity, and the connective-tissue
organization were evaluated at the same tissue sites as were the cellular
infiltration counts (Table II).
The histologic morphology of all of the tissues was evaluated by a veterinary
pathologist and physician (S.F.B.) and by one of the trained team members
(J.E.V.). Since specific phenotypic markers to distinguish between mononuclear
macrophages and lymphoid lineage cells were not used in the present study, the
generic and more conservative term "mononuclear cells" was used in
the analysis and in the description of the host response to all test
articles.
Statistical Methods
For each of the four response variables (cellularity, multinucleated giant
cells, vascularity, and degree of connective-tissue organization), ten fields
were scored on an integer scale of 0 to 3
(Table II). Averages of the ten
scores for each animal were analyzed statistically with use of a two-way
analysis of variance model (version 9, SAS; SAS Institute, Cary, North
Carolina)40. The
factors were the type of scaffold material (six levels, including the
autologous control graft plus the five biologic scaffold materials) and time
(seven levels). There were three animals for each time-treatment combination.
The data were reanalyzed with use of nonparametric methods based on ranks,
yielding results that were qualitatively the same.
All but one animal survived the surgical procedure without problems. Three
rats had development of age-related problems that required them to be killed
before the scheduled time. These four rats were replaced in the study to
maintain equal numbers in each group and subgroup.
Histopathologic Findings
The mean rounded scores for the three animals in each group by
time-treatment combination are presented in
Table III. For each response
variable, both main effects and the interaction were significant (p < 0.01)
in the analysis of variance.
The most intense response as judged by the number of cells at the implant
site over all time-intervals was observed with GraftJacket, Restore, and
CuffPatch, but the temporal appearance of the cell response differed for each
of the test articles. Multinucleated giant cells were present at some point
during the host response with GraftJacket, CuffPatch, and Permacol but were
never seen with Restore, TissueMend, or autologous tissue. The number of blood
vessels at the implant site was greatest with autologous muscle, Restore, and
CuffPatch, but the appearance of this vascular response differed with respect
to time for each of the devices. The connective-tissue organization at the
112-day end point was greatest and most similar to native tissue with the
Restore device.
Morphologic Findings
At two days, the grafts were surrounded by a moderate accumulation of
neutrophils. At four days of implantation, the neutrophil and mononuclear cell
populations increased in number for all grafts.
For the autologous abdominal wall tissue graft, there was progressive
necrosis of skeletal muscle fibers and an increased number of both neutrophils
and mononuclear cells that peaked at seven days
(Fig. 1-A). Tissue edema and
necrosis with complete loss of tissue and cellular architecture occurred by
fourteen days, at which time there was new collagenous connective tissue
deposition and prominent angiogenesis. At twenty-eight days, a moderate amount
of disorganized connective tissue was present at the implant site, consistent
with scar-tissue formation. Remnants of muscle fibers were scattered randomly
throughout the tissue, with a small number of mononuclear cells. The fifty-six
and 112-day time-points showed replacement of the autologous graft by dense,
poorly organized collagenous connective tissue and scattered islands of
adipose tissue (Fig. 1-B).
At seven days, the cellular response for GraftJacket consisted primarily of
dense accumulations of mononuclear cells, with occasional multinucleated giant
cells surrounding the graft site (Fig.
2-A). By fourteen days, the cellular infiltrate was dense and
consisted exclusively of mononuclear cells. The GraftJacket architecture was
disrupted at the edges with deposition of small amounts of new extracellular
matrix at fourteen days. By twenty-eight days, moderate angiogenesis, minimal
graft degradation, and a prominent mononuclear cellular infiltrate were
observed. By fifty-six days, the new host connective tissue at the edges of
the graft material was moderately dense, the cellular infiltrate had subsided,
and the majority of the graft material was unchanged from the appearance of
the original scaffold article. At 112 days after surgery, there was partial
degradation of the GraftJacket device and replacement with dense, partially
organized collagenous connective tissue
(Fig. 2-B).
For the Restore device, the cell population was mostly mononuclear in
morphology and was distributed throughout the graft material at seven days.
The individual layers of the Restore device were still distinct and
identifiable at seven days, but new host-derived extracellular matrix was
deposited between the layers of the original Restore device
(Fig. 3-A). By fourteen days,
there was abundant angiogenesis and extensive infiltration of the
extracellular matrix scaffold material by sheets of mononuclear cells. A loss
of the distinct ten-layer multilaminate device architecture occurred at
twenty-eight days, and only remnants of the Restore device were still
identifiable. By fifty-six days, islands of muscle cells and adipose
connective-tissue cells were found within the implant site and partially
organized connective tissue replaced almost the entirety of the Restore
device. By the 112-day time-point, the vascularity of the implant site was
still very prominent and the Restore device appeared to be completely degraded
and replaced with a mixture of organized muscle cells, collagenous connective
tissue, and small islands of adipose connective tissue
(Fig. 3-B).
The CuffPatch device showed an increased neutrophil population by seven
days, with a moderate amount of tissue edema that extended into the adjacent
native abdominal wall tissue (Fig.
4-A). There was minimal infiltration of cells beyond the edges of
the test article at fourteen days, and new host extracellular matrix and
fibrous connective tissue was deposited in the space immediately surrounding
the implant. Multinucleated giant cells were scattered throughout the
periphery of the implant site from fourteen days until the end of the study.
At twenty-eight days, neutrophils and mononuclear cells separated the
individual layers of the CuffPatch device in the central regions of the graft
site and the vascularity of the surrounding tissue was very prominent.
Scaffold degradation was apparent by fifty-six days, and a robust inflammatory
cell reaction, primarily mononuclear in nature, infiltrated the entirety of
the scaffold material. By 112 days, a typical foreign-body tissue response was
present, including the presence of multinucleated giant cells. Remnants of the
originally implanted biomaterial were still identifiable, with little
organization of new host connective tissue at the implant site
(Fig. 4-B).
For the TissueMend device, there was an absence of host cell invasion by
seven days, and small amounts of fibrous connective tissue were deposited at
the periphery of the device (Fig.
5-A). The vascularity at the interface between the native host
tissue and the TissueMend device was increased above that of the surrounding
normal tissue, and it remained increased for the entire 112-day study period.
By twenty-eight and fifty-six days after surgery, the fibrous capsule had
increased in thickness, with little infiltration of the TissueMend device by
host cells. By 112 days, there was degradation of scaffold material at the
edges of the device. Adipose connective tissue accumulated near the borders of
the TissueMend device, and a dense, highly organized connective tissue capsule
was present (Fig. 5-B).
For the Permacol device, poorly organized fibrous connective tissue
deposition was noted as early as four days. From fourteen to fifty-six days,
the fibrous connective tissue capsule around the device was the predominant
morphologic finding. Small numbers of mononuclear cells were present at the
edges of the scaffold material, and multinucleated giant cells were present
from seven days (Fig. 6-A)
until fifty-six days. By fifty-six and 112 days, the host cellular response
was minimal. There was almost no evidence of scaffold degradation at the
112-day time-point, and a thin, fibrous connective-tissue capsule surrounded
the device (Fig. 6-B).
We compared the host tissue response to five commercially available
extracellular matrix-based scaffold materials and autologous tissue in an
established rodent model of body-wall repair. The marketing literature for all
devices claims host acceptance and intact functionality. The host response in
the present study showed that there were differences in the amount and
temporal appearance of inflammatory cells, the morphologic structural
integrity of the devices over time, and the type of host tissue that either
replaced or surrounded the extracellular matrix-derived devices.
At the final time-interval (112 days), the remodeling outcome differed
markedly among the devices. The autologous control tissue showed typical
scar-tissue formation. The host tissue response to GraftJacket showed
replacement of the device with fibrous connective tissue and a persistent
low-grade chronic inflammatory response. The Restore device was replaced with
a mixture of muscle cells and organized connective tissue, a finding that is
consistent with an earlier report in which an eight-layer form of the small
intestinal submucosa material was used as a body-wall repair device in rat and
dog models33. The
CuffPatch device showed an accumulation of dense collagenous tissue and a
persistent foreign-body response at the 112-day time-point and showed a slower
remodeling process as compared with either the autologous control tissue or
the Restore device. The host response to TissueMend and Permacol was
consistent with the typical response to a nonresorbable foreign material; that
is, the response was characterized by low-grade chronic inflammation, minimal
scaffold degradation, and fibrous encapsulation.
The cellular component of the host inflammatory response, specifically, the
neutrophil and mononuclear cell populations, showed temporal and spatial
distribution differences between the scaffold materials. The intensity of the
cell response was greatest for the GraftJacket and Restore devices, especially
in the seven to fourteen-day period following implantation. The presence of
multinucleated giant cells, a cell type that is typically associated with a
foreign-body response, was observed at surgical sites in which GraftJacket,
CuffPatch, and Permacol were implanted. Of interest, the cellular response to
CuffPatch appeared to be predominately a neutrophilic-type response throughout
the entirety of the study. In contrast, the cellular response to GraftJacket
and Restore became primarily mononuclear in nature within seven days after
implantation. The classic paradigm of mammalian inflammation associates
neutrophils with an active, acute, or persistent host inflammatory
response41 and
associates mononuclear cells with a subacute to chronic type of inflammatory
response42-44.
Conventional knowledge suggests that mononuclear macrophages follow
neutrophils into a site of inflammation over time, phagocytose cellular debris
and foreign material, and finally exit from the site of
inflammation41,42.
This pattern of cellular response was observed for the autologous tissue-graft
control group. However, the cellular response to the naturally derived
scaffold materials investigated in the present study was not conventional and
differed for each device.
The host tissue vascular response was most prominent in association with
the Restore, CuffPatch, and GraftJacket devices. The tissue vascular response
may be a component of the host inflammatory reaction, or it may be
independently associated with the release of constituent growth factors as
part of the scaffold-degradation process. It has been shown that vascular
endothelial growth factor (VEGF), transforming growth factor beta
(TGF-ß), and basic fibroblast growth factor (bFGF) are incorporated
within the small intestinal submucosa material found in the Restore and
CuffPatch devices and that active forms of these factors survive the peracetic
acid-processing step and terminal sterilization
steps24,45-48.
These growth factors stimulate the proliferation and migration of cell
populations involved in inflammation, angiogenesis, vasculogenesis, and
fibrous-tissue deposition. The devices composed of extracellular matrix
material other than small intestinal submucosa may also retain growth-factor
activity following device preparation, but published studies to that effect
are lacking.
The evaluation of scaffold degradation in the present study was based on
the morphologic appearance of the implant site and our ability to recognize
intact devices. Because no molecular or radioactive labeling of the devices
was performed for a quantification of the rate of scaffold degradation, it is
possible that remnants of the remodeled scaffold materials remained at the
site and were incorporated into the new host tissue even when histologic
evaluation failed to recognize their presence. The only scaffold material for
which quantitative data exists for in vivo degradation is the non-crosslinked
form of small intestinal submucosa. Those studies used 14C -labeled
small intestinal submucosa materials and showed rapid and complete elimination
of the extracellular matrix scaffold within ninety days, with concomitant
replacement with host cells and new extracellular matrix
deposition49.
A recent study showed that the degradation products of extracellular matrix
have the potential for biologic activity such as chemoattraction for
endothelial cells, bone marrow-derived cells, and other cell
types23. Similarly,
antimicrobial activity has been reported for the degradation products of
extracellular matrix-derived scaffold
material25. The
above findings suggest that at least a portion of the host biologic response
to extracellular matrix-derived materials is associated with and/or caused by
the degradation products of the scaffold itself. Conversely, the lack of
degradation of a biologic scaffold material would logically suggest that such
downstream effects would not occur.
Fibrous encapsulation of implanted materials has long been associated with
the inability to remove these materials from the implant
site43,50.
Extracellular matrix scaffolds that are processed in such a way as to minimize
degradation, for example, through chemical crosslinking
methodologies51-54,
are more likely to be associated with fibrous encapsulation and chronic
inflammation38,55,56.
There was a distinct fibrous capsule around the Permacol and TissueMend
devices. These devices also showed the slowest rate of scaffold degradation
over the course of 112 days. Although TissueMend does not include chemical
crosslinking as a processing step, the proprietary methodology of making the
final product may be related to its relatively slow rate of degradation.
In conclusion, this head-to-head comparison of the clinically available
extracellular matrix scaffold materials in an animal model indicates that the
five scaffold devices currently used for musculotendinous repair are distinct
with regard to the host tissue response. All of these devices are composed of
mammalian extracellular matrix but differ in terms of their processing
methods, species of origin, and tissue of origin. A limitation of the present
study is that it was conducted in a rodent model, which may or may not be
predictive of the response seen in humans. Additional work is needed to better
understand the cellular mechanisms of biologic scaffold remodeling and to
correlate such findings with the methods by which the biomaterials are
processed and with the eventual clinical outcome.
A table showing the Food and Drug Administration-approved clinical uses for
each extracellular matrix device 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). ?