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.