The production of 14C-labeled extracellular matrix derived from
small intestinal submucosa and the surgical procedure performed as part of
this study were approved by the University of Pittsburgh Radiation Safety
Committee and the Institutional Animal Care and Use Committee of the
University of Pittsburgh. The animal care complied with the National
Institutes of Health Guidelines for the Care and Use of Laboratory
Animals.
Preparation of 14C-Labeled Small Intestinal Submucosa
Extracellular Matrix
The methods for labeling the small intestinal submucosa extracellular
matrix with 14C have been reported
previously19.
Briefly, piglets were given weekly intravenous injections of 10 µCi of
14C-labeled proline (256 mCi/mmol, 50 µCi/mL; Amersham Life
Science, Piscataway, New Jersey) beginning at three weeks of age and
continuing until the time the animal was killed. At approximately twenty-six
weeks of age, the animals were killed and the small intestine was harvested.
The small intestine was mechanically abraded to remove the tunica muscularis
externa and the majority of the tunica mucosa. The remaining tunica submucosa
and basilar portion of the tunica mucosa was then disinfected and
decellularized in a 0.1% peracetic acid solution followed by two rinses each
in phosphate-buffered saline solution and deionized
water22.
To produce each device for the Achilles tendon repair, a ten-layer
construct of the hydrated sheets of 14C-labeled small intestinal
submucosa extracellular matrix was created such that the longitudinal axis of
each sheet of the small intestinal submucosa was oriented in the same
direction. The small intestinal submucosa has a preferred collagen fiber
orientation along the long axis of the small
intestine23;
therefore, creating the construct in this uniaxial orientation provided
increased strength in the direction aligned with the repaired tendon. The
ten-layer construct was then laminated by a vacuum-pressing
technique22. The
construct was placed between two perforated stainless-steel sheets, and the
stainless-steel plates were placed between sheets of sterile gauze. The entire
construct was then sealed in vacuum bagging and was subjected to a vacuum of
710 to 735 mmHg (94.8 to 98.2 kPa) for approximately eight hours. The
multilaminate device was cut into 2 × 3-cm sheets, with the preferred
fiber direction corresponding to the 2-cm axis. The final construct was
terminally sterilized by exposure to ethylene oxide.
Study Design
Twelve adult female mongrel dogs weighing approximately 20 kg were divided
into six groups of two dogs each. All dogs were subjected to segmental
resection of 1.5 cm of the Achilles tendon, and the defect was repaired with
the bioscaffold composed of 14C-labeledsmall intestinal submucosa
extracellular matrix. One group of animals was killed at each of the following
time-points: three, seven, fourteen, twenty-eight, sixty, and ninety days
after surgery.
Surgical Procedure
A surgical plane of anesthesia was produced in each dog by induction with
12 to 25 mg/kg of sodium thiopental administered intravenously followed by
intubation and maintenance with 2% to 3% isoflurane. The animals were prepared
for surgery by shaving the surgical site and scrubbing with Betadine
(povidone-iodine) solution. An antibiotic (cephalexin) was administered
intramuscularly preoperatively and then postoperatively twice a day for seven
days. For each dog, the Achilles tendon of a randomly selected hind limb was
dissected free of surrounding tissue and a 1.5-cm segment was excised from the
mid-tendinous region. The tendon defect was repaired with a 2 × 3-cm
graft of the 14C-labeled scaffold folded accordion style, such that
2 to 3 mm of the 2-cm length overlapped each end of the tendon defect, with
use of 4-0 Prolene (polypropylene) suture to mark the ends of the defect
(Fig. 1). The paratenon was
sutured with 4-0 PDS (polydioxanone) suture, and the skin incision was closed
with use of standard surgical technique. The animals were placed in a tube
splint (a modified Robert Jones bandage) to allow immediate partial
weight-bearing without allowing the tendon unit to stretch. The splint was
removed after twenty-eight days, and the dogs were allowed to walk freely
without external support.
Sample Collection
At regular time-intervals after surgery, urine, blood, and fecal samples
were collected to determine the route of elimination of the degraded
14C-labeled materials from the body. At the time the animals were
killed, the remodeled graft and the contralateral, uninvolved control Achilles
tendon were harvested for histological examination and quantification of the
14C concentration within the tissue. The specimen prepared for
histological analysis contained remodeled normal tendon and remodeled
extracellular matrix so that the transition between the two tissues could be
evaluated. The specimen for 14C analysis was taken from the middle
of the remodeled graft. Tissue samples were collected from the skin, skeletal
muscle, mesenteric fat, spleen, liver, kidney, pancreas, lymph nodes, lung,
heart, and brain to determine whether 14C had collected in these
organs.
14C Analysis
The radioactivity in each sample was measured by liquid scintillation
counting with use of a B-counter (model LS 1800; Beckman Coulter, Somerset,
New Jersey). For urine, 0.2 mL of urine was added directly to 10 mL of
scintillation fluid (Ultima Gold; PerkinElmer, Boston, Massachusetts). The
radioactivity in the urine was reported as counts per minute per milliliter
(cpm/mL). For the extracellular matrix graft at time-zero, the remodeled
tendon, and all other harvested tissues, approximately 80 mg of tissue was
incubated with 1 mL of tissue solubilizer (Solvable; Packard Instruments,
Meriden, Connecticut) at 50°C for two to four hours. Ten milliliters of
scintillation fluid was then added to each sample, and the radioactivity was
determined. The tissue radioactivity was reported as counts per minute per
gram (cpm/g).
Blood samples were incubated with a 1:1 mixture of tissue solubilizer to
isopropanol (Fisher Scientific, Pittsburgh, Pennsylvania) at 60°C for
thirty minutes. The samples were then treated with 30%
H2O2 (Spectrum, Gardena, California), to quench
endogenous peroxidase activity, and were incubated at 60°C for an
additional thirty minutes. Fifteen milliliters of scintillation fluid was
added to each sample, and the radioactivity was measured. The blood
radioactivity was reported as counts per minute per milliliter (cpm/mL). To
measure the radioactivity in a fecal sample, approximately 20 mg of feces was
rehydrated with 0.1 mL of water for thirty minutes at room temperature. One
milliliter of tissue solubilizer was added to each sample, and the samples
were incubated in a 50°C oven for one to two hours. Isopropanol (0.05 mL)
was added before the final two-hour incubation at 50°C. Peroxidases were
quenched with 0.2 mL of 30% H2O2, and 10 mL of
scintillation fluid was added. The radioactivity of the feces was reported as
counts per minute per gram (cpm/g).
Histological Analysis
A portion of each tendon was harvested and trimmed to include the remodeled
extracellular matrix graft and the adjacent native tendon proximal and distal
to the remodeled graft. The tissue was fixed in 10% neutral buffered formalin,
sectioned longitudinally, and was stained both with hematoxylin and eosin and
with Masson trichrome for histological examination. The cellularity of the
remodeled graft was assessed by obtaining five photomicrographs from each
section at 40× magnification for subsequent counting with use of MetaVue
software (Universal Imaging, Downingtown, Pennsylvania). The cells were
classified as neutrophils or mononuclear cells. The data are presented as the
average number of cells per high-power (40×) field. For statistical
comparisons, the Student t test was used to compare differences between groups
and significance was set at a p value of <0.05.
Surgical Outcome
All animals recovered well from surgery, and they tolerated the splinting
for one month. After removal of the splint, all animals returned to a normal
gait. Gross evaluation of the repaired tendons after the animals were killed
showed no evidence of tendon rupture at any time during the postoperative
period. By visual inspection, there did not appear to be any change in the
total tendon length or the length of the repair site.
14C Analysis
The values for 14C concentration of the remodeling extracellular
matrix tissue for each dog are listed, and the average values at each
time-point are plotted in Figure
2. Approximately 10% of the scaffold material had been degraded
and removed from the site of remodeling by as early as three days after
surgery. By fourteen days, approximately 20% of the scaffold material had been
degraded, and, at twenty-eight days, approximately 60% of the scaffold
material had been degraded. By sixty days after surgery, the amount of
14C in the remodeled tissue was equal to background levels
(<8%), indicating complete scaffold degradation and removal from the site
of remodeling.
The only other tissues in which 14C activity could be detected
were blood and urine. The blood showed positive 14C activity at
three and seven days after surgery. The urine showed 14C activity
at three, seven, and fourteen days after surgery. All other tissue samples
were negative (i.e., below background values) for 14C activity at
all time-points.
Histological Analysis
Histological examination of the extracellular matrix graft prior to
implantation showed a laminate structure of dense, organized, collagenous
tissue with no cellularity (Fig.
3).
Tissues harvested from the remodeling graft and tendon showed that, at
three days after surgery, the acellular scaffold material had been infiltrated
with a large number of host inflammatory cells characterized by an
approximately equal number of neutrophils and mononuclear cells
(Table I). Early evidence for
scaffold degradation included the separation of the individual sheets within
the multilaminate structure by infiltrating host cells and a loss of distinct
scaffold architecture at the periphery of the graft material. There was no
histological evidence for deposition of new host extracellular matrix on the
basis of the distinct margins of the graft and the cut end of the native
tendon.
By seven days (Fig. 4), the
cellular infiltrate had increased in amount and consisted almost exclusively
of mononuclear cells (Table I).
A moderate degree of scaffold degradation was present, and the host-cell
infiltrate had progressed from the periphery of the graft material to the
center of the graft material. New host extracellular matrix was present at the
remodeling site and was represented by the presence of amorphous connective
tissue between the native tissue and the graft material.
By fourteen days (Fig. 5),
the entire scaffold material was infiltrated with host cells that were
primarily mononuclear in morphology with only a few scattered neutrophils
being present. This time-point showed the greatest cellularity over the course
of remodeling with an average (and standard deviation) of 271 ± 133
cells per high-power (40×) field
(Table I). Graft degradation
was characterized by separation of the various layers of the multilaminate
sheet and loss of a distinct boundary between newly deposited host
extracellular matrix and the original graft material. Deposition of new
host-derived extracellular matrix was present, especially at the periphery of
the graft. The distinct line of demarcation between the scaffold material and
the cut end of the native Achilles tendon to which it was attached was no
longer evident by fourteen days.
By twenty-eight days (Fig.
6), there was a decrease in the cellularity similar to the numbers
observed at seven days after implantation
(Table I). The mononuclear
cells were still abundant and were uniformly distributed throughout the
remodeled extracellular matrix graft. No neutrophils were observed. There was
loss of almost all morphologic evidence of the graft material, which was
replaced by a relatively homogeneous deposition of new host-derived
extracellular matrix material. The remodeled extracellular matrix was
beginning to show regions of organization by this time-point.
At sixty days (Fig. 7),
there was replacement of the graft material by organized, aligned host-derived
extracellular matrix and spindle cells consistent with the morphologic
appearance of fibroblasts. The total cellularity was not different from the
cellularity at twenty-eight days (Table
I). The remodeled graft was replaced by organized connective
tissue, and the site could only be identified from the native tissue by the
presence of the Prolene suture.
The ninety-day tissue samples (Fig.
8) showed a slight decrease in the number of spindle cells present
within the graft material compared with the sixty-day sample, and the
cellularity was no different from the normal Achilles tendon
(Table I). The collagen fiber
organization and vascularity were also qualitatively similar to those of
normal tendon tissue.
The results of this study show that extracellular matrix derived from small
intestinal submucosa, when used as a biologic scaffold for Achilles tendon
reconstruction in this canine model, is rapidly degraded after implantation,
with approximately 60% of the mass degraded and resorbed within four weeks.
Degradation of the scaffold appears to be complete by three months. These
findings are almost identical to the degradation rate of small intestinal
submucosa extracellular matrix when this biologic scaffold was used for
reconstruction of the urinary
bladder19. The
resorbed 14C-labeled degradation products in the present study were
eliminated from the body primarily by urinary excretion. No detectable
14C was found in any of the parenchymal organs that were
examined.
The nonlinear temporal degradation of the scaffold was associated with the
extent and distribution of the host cellular infiltrate. At the time of
implantation, no cells were present within the scaffold. As cells infiltrated
the graft, the rate of scaffold degradation increased. By four weeks after
surgery, the cell infiltrate had lessened and the rate of scaffold degradation
had begun to diminish. The distinctive laminated architecture of the small
intestinal submucosa extracellular matrix graft was no longer distinguishable
by twenty-eight days, and the remodeled scaffold showed a relatively uniform
accumulation of homogenous collagenous connective tissue.
The Achilles tendon is subjected to some of the highest stresses of any
tendon in the
body24-27.
It would therefore seem logical that an extracellular matrix scaffold that is
subjected to rapid degradation, such as small intestinal submucosa
extracellular matrix, would be at risk of failure when used as a repair device
for the Achilles tendon under physiologic loading, especially during the first
four to eight weeks after surgery. In fact, this concern has been the
rationale for chemically cross-linking many extracellular matrix scaffolds
including one commercially available device derived from small intestinal
submucosa extracellular matrix (CuffPatch;
Arthrotek)28-30.
In the present study, a tube splint that allowed for partial weight-bearing
was utilized for the first month after implantation to prevent failure due to
suture pull-out while still allowing the extracellular matrix scaffold to bear
load. The application of load early in the remodeling process has been shown
to be important for constructive
tissue-remodeling17.
After removal of the splint in the present study, the remodeled scaffoldwas
sufficiently strong to withstand unrestricted cage activity in this animal
model without evidence of rupture. The remodeled scaffold showed organized,
aligned collagenous tissue after removal of the tube splint at twenty-eight
days. Although mechanical testing was not performed in this study, a previous
study with use of the same canine model of Achilles tendon repair showed that
the strength of the remodeled Achilles tendon exceeded the strength of the
insertion to the gastrocnemius muscle and the calcaneus by twelve weeks after
repair with small intestinal submucosa extracellular
matrix16.
Recent studies have suggested that degradation of an extracellular matrix
scaffold may be an essential component of a constructive remodeling response
as opposed to scar-tissue
deposition31-34.
Low-molecular-weight peptides formed during the degradation of small
intestinal submucosa extracellular matrix have been shown to have
chemoattractant properties for several cell types in vitro and angiogenic
potential in
vivo32. Chimeric
mouse models, including a model in which small intestinal submucosa
extracellular matrix was used to repair the Achilles tendon, showed that bone
marrow-derived cells are recruited to the site of healing and that they
participate in the long-term remodeling of the
scaffold31,34.
In addition, in vitro studies have shown that degradation products of
extracellular matrix scaffolds have antibacterial
properties33, but,
in the absence of degradation, the extracellular matrix scaffolds support
bacterial growth35.
It is plausible that chemoattractance by degradation products contributed to
the recruitment of host cells and, ultimately, to tendon remodeling. Stated
differently, degradation of an extracellular matrix scaffold may be a
requisite process with bioactive consequences that contribute to the overall
remodeling events. Inhibition of scaffold degradation by chemically
cross-linking the extracellular matrix may decrease or eliminate the
beneficial effects of extracellular matrix degradation products.
A limitation of the present study is the small number of animals (two) that
were evaluated at each time-point. The low numbers were a result of practical
considerations, including the cost of the study and the complexity involved in
producing the small intestinal submucosa extracellular matrix labeled with
14C. Despite these limitations, this study showed rapid degradation
of a small intestinal submucosa extracellular matrix scaffold used for the
repair of a musculotendinous tissue, an application for which small intestinal
submucosa extracellular matrix is currently in clinical use. This study also
shows that, as degradation occurs, the graft remodels into dense collagen-rich
connective tissue with an organization, cellularity, and vascularity similar
to that of native tendon tissue. ?