No defect recurrences were observed throughout the course of the study. One sheep developed an infection at the implanted site and was euthanized and replaced by a reserve animal. Within the first month after surgery, abdominal swelling was observed in eight sheep that otherwise showed good general health status. The swelling was likely due to seroma formation provoked by the rather extensive dissection needed to fit four implants on each animal. Five of these sheep showed hyperthermia and received antibiotics for 4 days. Spontaneous regression was observed, except for one animal on which punctures were performed under sterile conditions. After these punctures, spontaneous regression was observed.
At all time points, the macroscopic evaluations showed no local adverse effects for either the test or control meshes, and no biologically significant differences were ever observed macroscopically. For both test and control, the mesh-tissue complex showed early neovascularization and the meshes were well integrated into fibrous connective tissue. Some differences between individual animals were noted at all time points. In general, the control mesh was easier to locate due to its stiffness as compared to the test mesh, where the mesh-tissue complex was difficult to differentiate from the intact abdominal wall upon palpation of the sites. This was more evident at the 24- and 36-month sacrifices as the polypropylene control mesh was stiff, yet not well integrated and the tissue layers were easily separated from the mesh following explantation. As expected, the control mesh never showed any signs of degradation whereas the first fiber of the test meshes was completely degraded after 4 months and all test meshes were macroscopically undetectable at 36 months.
There were no histological signs, at any time, of residual infection or necrosis for either test or control meshes. At 4 months, the control mesh showed no signs of alteration, and elicited a chronic inflammatory reaction (Fig. 2) characterized by a moderate grade of phagocytotic cells infiltrating the mesh, and a slight grade of neutrophils, plasma cells and lymphocytes. A moderate grade of fibroplasia was observed peripherally and a large amount of mature collagen of marked grade was observed between the fibers, but also surrounding the mesh.
After 4 months, the fast-degrading fiber of the test mesh was histologically absent. The slow-degrading multifilament fibers of the test mesh were surrounded by a moderate grade of fibrous tissue and induced an inflammatory reaction qualitatively and quantitatively similar to that observed with the control mesh, except for a higher amount of collagen deposition with a more variable maturity and an occasionally greater lymphocytic reaction (Fig. 3a). A moderate degree of neovessel formation could be observed in the surrounding tissue as well as adjacent to the mesh fibers. A small proportion of the test sites showed slight to moderate grade of interfascicular fatty tissue deposition, which was considered to be incidental (Fig. 3b).
The histological appearance of both test and control meshes was similar at 4 and 9 months. At 9 months, test sites were still characterized by a higher amount of collagen deposition, ranging from marked to severe, leading to thicker connective tissue, infiltrating and surrounding the mesh (Fig. 4a). Histological analysis of the collagen types showed that the test implants were associated with a higher rate of connective tissue maturation when compared to control implants after 9 months. The quality of the collagen was deemed closer to the native abdominal wall within the test implant sites.
At 15 and 24 months, the control mesh showed a slight chronic inflammatory reaction. A moderate grade of colonization and slight grade of encapsulation was evident, along with a marked grade of integration of the fibers (Fig. 4b). The test mesh induced an inflammatory reaction qualitatively and quantitatively higher than that of the control mesh, due mainly to a higher recruitment of phagocytotic cells around and between the multifilaments (Fig. 4c). This is part of the degradation process  as the fibers constituting the multifilament displayed evidence of degradation under polarized light. Collagen deposition seemed to be an ongoing process 15 months post implantation.
The overall thickness of mature collagen deposition was higher (score 2.8) than in the control group (score 2) at 24 months. The fibers constituting the multifilaments of the test mesh displayed an advanced stage of degradation under polarized light, and fibers of decreased size were found commonly in the cytoplasm of macrophages and giant cells indicating an active phagocytotic process (Fig. 5a).
The test mesh was fully degraded at 36 months and replaced by a connective tissue of variable thickness, and only a few implant residues were observed (Fig. 5b). The control mesh showed no signs of degradation at 36 months after implantation. The inflammatory response to the test mesh had decreased significantly between the 24 and 36 months time points and was clearly lower compared to the control group at 36 months. Only a few macrophages and giant cells showing the intracytoplasmic presence of material debris could be observed. The general connective tissue layer formed was thicker around the test mesh as compared to the control.
Scanning electron microscopy
The thickness of the native abdominal wall was approximately 5 mm, with normal layers of muscle, fat and connective tissue. The control mesh showed a wall thickness of approximately 1.3 mm after 9 months, whereas the test specimen revealed a newly formed abdominal wall of approximately 6 mm. At 4 and 9 months, both test and control specimens showed a well-integrated mesh without signs of encapsulation. The collagen surrounding the filaments was mature with marked signs of remodeling and maturity. These findings were consistent at 24 and 36 months.
In the early phase of wound healing, granulation tissue rich in collagen type III, which is characterized by weak tensile properties, is deposited. Later in the wound healing process collagen type III is replaced by collagen type I characterized by increased tensile properties [21, 22]. In an attempt to determine the ratio between collagen types I and III, selected histology sections from the 9-month observation period were stained according to the method described by Junquiera . The result revealed a slightly higher ratio of collagen type I at the test sites associated with a higher rate of connective tissue maturation when compared to the control implants.
Based on the above results, a more thorough histomorphometric analysis of the collagen ratio in some of the slides originating from 15, 24 and 36 months observations was performed.
Specimens were taken only from animals carrying both test and control meshes in order to compensate for individual variability.
In both histology and SEM analysis, the test sites displayed a gradual increase in collagen content within the extracellular matrix between 15 and 36 months post implantation (Fig. 6a). This is in sharp contrast to the control sites, which showed a gradual decrease in the collagen content within the extracellular matrix formed around and within the control mesh. One explanation for this could be the fat tissue that progressively infiltrated the extracellular matrix over time.
For the test sites the collagen was found to integrate well within the mesh after 24 months and could also be found between individual fiber bundles as well as surrounding the mesh. At 36 months the mesh was no longer visible and the resorbed fiber bundles were fully replaced by new collagen-rich tissue, which could explain the increase in collagen content found within the extracellular matrix.
The ratio of collagen I/III within the test sites increased over time (Fig. 6b), indicating a continuous remodeling of the ingrown collagen towards a strong connective tissue. The control site showed little or no remodeling after 15 months of implantation, with less collagen type I deposited within the extracellular matrix surrounding the control mesh as opposed to the test site.