2. Materials and Methods
2.1. Ethical Considerations
2.3. Physico-Chemical Characterization of the Membranes
2.4. Animal Characterization
2.5. Surgical Procedures
2.6. Sample Obtention
2.7. Microscopic Evaluation
2.7.1. Histological Description
2.7.2. Semiquantitative Evaluation of Inflammatory Infiltrate
- The quantity and distribution of inflammatory cells present at the tissue–material interface. The neutrophils, lymphocytes, plasma cells, and macrophages were classified as: absent (score zero), rare or 1–5 per high-powered (400×) field (phf) (score 1), 5–10 phf (score 2), heavy infiltrate (score 3), and packed (score 4). The multinucleated cells were classified as: absent (score zero), 1–2 per high-powered (400×) field (phf) (score 1), 3–5 phf (score 2), heavy infiltrate (score 3), and sheets (score 4).
- The inflammatory response parameters (neovascularization, the degree of fibrosis, and fatty infiltrate.
- The presence of necrosis.
3.1. Scanning Electron Microscopy (SEM)
3.2. Fourier Transform Infrared Spectrometer (FTIR)
3.3. In Vivo Response to Membranes and Macroscopy Results
3.4. Descriptive Histological Analysis
3.4.1. One Week Post-Implantation
- Group 1: In G1, we observed the presence of the membrane with a fibrillar aspect and permeated with mesenchymal cells (fibroblasts), mononuclear inflammatory cells and scarce polymorphonuclear cells, and abundant multinucleated giant cells (MNGCs), both in the periphery and permeating the membrane. Some MNGCs had material in their interior. Surrounding the membrane was a narrow band of granulation reaction of predominantly mononuclear inflammatory infiltrate (Figure 6A,B).
- Group 2: In all animals in this group, it was not possible to visualize the membrane: a dense band of connective tissue containing an intense mixed inflammatory infiltrate was observed (Figure 6C,D). The membrane appeared not to have been incorporated into the underlying tissues and detached during histological processing. It was possible to observe the contour or virtual space that the membrane occupied.
- Group 3: In all animals in this group, no membrane was identified, similarly to Group 1. The membrane appeared not to have been incorporated into the underlying tissues and detached during histological processing. Additionally, in the contour, it was possible to observe connective tissue containing moderate mixed inflammatory infiltrate (Figure 6E,F).
- Group 4: In this group, we observed the presence of a thick membrane (M) with a fibrillar aspect, covered by a narrow band of connective tissue with a mild to moderate granulation reaction. Multinucleated giant cells were observed in its periphery, sometimes containing material inside. Permeating the membrane, there was the presence of fusiform and starred mesenchymal cells (Figure 6G,H).
- Group 5: The presence of a thick membrane with a spherical and tubular aspect, peripherally interspersed with delicate connective tissue bundles, was observed. We noticed cell adherence on the spherical structures of the membranes and a few multinucleated giant cells. In the membrane, we observed connective tissue with a mild inflammatory infiltrate (Figure 6I,J).
- Group 6: The dermis was lined by orthokeratinized stratified squamous epithelium exhibiting areas of extensive keratinization and scar hyperplasia; underlying fibrous connective tissue was observed with a focal area of intense mixed inflammation and collagen and muscle fibrous connective tissue with focal areas of intense mixed inflammation and organized collagen and muscle fibers (Figure 6K,L).
3.4.2. Three Weeks Post-Implantation
- Group 1: Membranes with a fibrillar appearance and permeating mesenchymal cells (fibroblasts), mononuclear inflammatory cells, and scarce polymorphs were observed, along with abundant multinucleated giant cells (MNGCs), both at the periphery and permeating the membrane, containing content. Some MNGCs displayed material inside. Integrated in the membrane was a delicate connective tissue with a few inflammatory cells (Figure 7A,B).
- Group 2: In G2, a rectilinear, homogeneous, and matte membrane appearing “loose” in almost the whole sample, without adherence to the connective tissue, was observed. In its periphery, multinucleated giant cells and mononuclear cells were trying to break through the membrane. A dense band of the granulation reaction in the connective tissue was subjacent (Figure 7C,D).
- Group 3: In this group, the membrane was rectilinear, homogeneous, matte, and “loose,” without adherence to the connective tissue. Multinucleated giant cells and mononuclear cells were present in its periphery, similarly to Group 2. In proximity to the membrane, there was connective tissue with a moderate mixed inflammatory infiltrate (Figure 7E,F).
- Group 4: We observed the presence of a thick fibrillar membrane permeated by sparse mesenchymal cells with a fusiform and stellate appearance. In the periphery, there was a narrow band of connective tissue with the granulation reaction, composed of a small amount of mononuclear inflammatory infiltrate with a predominance of macrophages. Multinucleated giant cells in its periphery were observed (Figure 7G,H).
- Group 5: A thick membrane with a spherical and tubular aspect interspersed with delicate connective tissue bundles was observed; cellular adherence on its surface was observed in some “spheres” of the membrane with few CGMNs. We observed connective tissue with mild inflammatory infiltrate integrated into the membrane (Figure 7I,J).
- Group 6: In the incision area, a reorganization of fibrous and muscular tissues interspersed with a moderate mononuclear inflammatory infiltrate was noted (Figure 7K,L).
3.4.3. Six Weeks Post-Implantation
- Group 1: In this group, it was observed that, six weeks after implantation, the membrane was interspersed with loosely arranged connective tissue and multinucleated giant cells with contents inside. The membrane was present in all animals (Figure 8A,B). The membrane, despite being permeated by a cell population and connective tissue, maintained its scaffold.
- Group 2: The presence of a rectilinear and homogeneous membrane with a microscopic matte appearance and without adherence to the adjacent connective tissue was observed. In the connective tissue surrounding the membrane, a granulation reaction with intense mononuclear infiltration also was observed, in addition to the presence of multinucleated giant cells in the periphery of the membrane (Figure 8C,D).
- Group 3: The membrane of G3 had a matte, rectilinear, and homogeneous aspect. Microscopically, it looked partially loose, without adherence to the adjacent connective tissue, limited by mild multinucleated giant cells and mononuclear cells. In the adjacent membrane, connective tissue with inflammatory cells was present (Figure 8E,F).
- Group 4: A thick membrane with a fibrillar aspect containing in its periphery a narrow range of the granulation reaction and multinucleated giant cells with content inside was observed. It is possible that the membrane was permeated by mesenchymal cells with a fusiform and stellate aspect (Figure 8G,H).
- Group 5: In this group, we observed a membrane composed of multiple structures with a clear basophilic spheroidal appearance bounded by multinucleated giant cells and macrophages. Fibrocellular connective tissue permeated the spheres (Figure 8I,J).
- Group 6: In this group, organized connective tissue with sparse inflammatory cells was observed. Adipose, muscular, and glandular tissues were present in the region (Figure 8K,L).
3.4.4. Twelve Weeks Post-Implantation
- Group 1: In this group, the membrane was surrounded by loosely arranged connective tissue with scant inflammatory cells and giant cells in the periphery. At the highest enlargement of the membrane, it was observed that multinucleated giant cells in the membrane presented contents inside, and mesenchymal cells were present permeating the membrane (Figure 9A,B). As in the case at six weeks, the membrane, although permeated by a cell population and connective tissue, had maintained its framework.
- Group 2: The membrane appeared rectilinear, intact, homogeneous, and, with a matte aspect, “loose”, without adherence to the connective tissue. In its periphery was observed the presence of multinucleated giant cells and mononuclear cells inside the membrane. A band of the granulation reaction was found underneath the membrane (Figure 9C,D).
- Group 3: The membrane was rectilinear, intact, homogeneous, matte, and “loose”, with no adherence to the connective tissue. In its periphery, the presence of multinucleated giant cells and a few mononuclear cells were observed. In proximity to the membrane, connective tissue with a few inflammatory cells was noted (Figure 9E,F).
- Group 4: In this group, the membrane had a fibrillar aspect and, contained in the periphery, a sparse mononuclear inflammatory infiltrate, and a large number of multinucleated giant cells were observed. It was possible to observe, at the highest magnification, the membrane permeated by mesenchymal cells with a fusiform and striated aspect and, in the periphery, multinucleated giant cells with content inside (Figure 9G,H).
- Group 5: The presence of a few membrane fragments interspersed with a band of connective tissue exhibiting granulation reaction was observed. From the details at the higher magnification, it was possible to observe membrane residues interspersed with the granulation reaction and multinucleated giant cells (Figure 9I,J).
- Group 6: In this group, well-organized tissue with scarce inflammatory cells and a normal healing appearance was observed for the 12-week period (Figure 9K,L).
3.5. Semiquantitative Histological Analysis of Local Biological Effect of Implanted Membranes: ISO 10993-6: 2016/Part 6/Annex E
3.5.1. PMN Cells
3.5.3. Plasma Cells
3.5.5. Giant Cells
Institutional Review Board Statement
Data Availability Statement
Conflicts of Interest
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