Figure 1: Images of test and control articles- A) The image
shows the clinically relevant CIED model used for the surgical implants.
B) The experimental group enclosed CIEDs in a CanGaroo ECM Envelope like
the one pictured here.
Upon closing the incision sites, the animals were allowed to recover and
were returned to standard individual housing. The incision sites were
continually monitored over the course of the study for any abnormalities
or incomplete wound healing. Animals were individually housed in
stainless steel cages/polycarbonate cages with slatted floors in
environmental conditions of temperature, relative humidity, and
photo-period according to NIH recommendations for this species as
reported in the “Guide for the Care and Use of Laboratory Animals”.
Potable water, certified commercial feed, and environmental enrichment
were supplied until animals were sacrificed at scheduled time points.
One animal receiving a CIED with CanGaroo ECM Envelope was sacrificed
and evaluated at 2 weeks post implant (the time point for skin incision
healing) to provide a histological starting point for expected ECM
envelope resorption and remodeling, which is designed to occur naturally
over time in the pocket. For the remaining animals in each group, four
implants were evaluated at week 13, and six were evaluated at 26 weeks.
At each time point, rabbits were humanely euthanized. Implant sites were
then opened, observed macroscopically, and then processed for
histopathology.
For macroscopic observations, the outer capsule surrounding the CIED at
each implant site was surgically exposed and photographed by removing
the layers of epidermis and dermis. Evidence of normal ECM envelope
resorption and constructive remodeling was documented. Presence of fluid
and capsule thickening or discoloration were also noted. Before and upon
capsule dissection, the surgeon recorded any movement or flipping of the
CIED from its original implanted position. The CIED was then removed,
and the full capsule was excised and marked with tissue dye for
orientation. The tissue was fixed in formalin for histopathological
analysis.
Histopathology and Capsule MeasurementsTwo (2) tissue pieces 1cm in length were excised for histology from the
middle and dorsal corner of each capsule. Samples were processed by
standard histopathology techniques including paraffin embedding,
sectioning (two serial sections at each sample midpoint), and staining
(one section per stain with hematoxylin and eosin [H&E] and
Trichrome). Slides were evaluated by a blinded veterinary pathologist
for local tissue responses, envelope resorption, and fibrotic tissue
formation. This analysis scored the appearance of mineralization,
necrosis, neovascularization, and fibrosis on an established scale of 0
to 4 (0 meaning absent and 4 meaning marked presence).
For assessment of CanGaroo ECM Envelope resorption which directly
correlates with remodeling, a baseline estimation of envelope area was
made in the slides from an initial 2 week time point. Subsequent
measurements of remnant product in the capsular tissue were made at 13
and 26 weeks and standardized to the 2 week time point. In addition to
resorption, thickness measurements of the tissue capsule were made at
three representative places from each individual H&E and trichrome
slide of each tissue sample, excluding remnant envelope material in the
measurements. The average of these measurements was used to estimate
fibrotic capsule thickness from every individual implant site and
subsequently averaged for each experimental group.
Results: Gross Necropsy Observations :
Upon examination of the subcutaneous pocket, normal, mild amounts of
fluid from surgical wound healing were present in both implant groups at
13 weeks. Fluid dissipated and became absent by the week 26 time point.
Connective tissue bands, associated with a normal subcutaneous response
to implants, were observed in both groups at the 13 and 26 week time
points with the tissue thickness increasing with implant duration.
Compared to intact envelope material and related tissue discoloration
found in the CIED with CanGaroo ECM Envelope group at 2 weeks, clear and
steady progress was seen in the tissue remodeling of the envelope by 13
and 26 weeks. This remodeling was accompanied by an increased level of
vascularization over time that appeared greater than levels found in
tissue surrounding the CIED without envelope group at 13 and 26 weeks(Figure 2) .
This connective tissue secured cardiac devices in place in the
subcutaneous pocket. However, CIEDs were highly prone to movement within
the pocket because of the normal, instinctive movement of the rabbits,
including rolling onto the implant site or directly rubbing their dorsal
side on the cage. Also, CIED leads were absent in this model, which
usually restrict movement in the subcutaneous space and further
stabilize the implants. This vulnerability lead to a high incidence of
device flipping within the pocket. During necropsy, 50% or five (5) out
of 10 total implants were documented to flip 180° cranially within the
pocket in the absence of an envelope. Only a single incidence of
flipping out of 10 implants was observed at either the 13 or 26 week end
points in the presence of CanGaroo ECM Envelopes.