Porcine Dermis and Pericardium-Based, Non–Cross-Linked Materials Induce Multinucleated Giant Cells After Their In Vivo Implantation: A Physiological Reaction?
The present study analyzed the tissue reaction to 2 novel porcine-derived collagen materials: pericardium versus dermis. By means of the subcutaneous implantation model in mice, the tissue reactions were investigated at 5 time points: 3, 10, 15, 30, and 60 days after implantation. Histologic, histochemical, immunhistologic, and histomorphometric analysis methodologies were applied. The dermis-derived material underwent an early degradation while inducing mononuclear cells together with some multinucleated giant cells and mild vascularization. The pericardium-derived membrane induced 2 different cellular tissue reactions. The compact surface induced mononuclear cells and multinucleated giant cells, and underwent a complete degradation until day 30. The spongy surface of the membrane induced mainly mononuclear cells, and served as a stable barrier membrane for up to 60 days. No transmembranous vascularization was observed within the spongy material surface layer. The present data demonstrate the diversity of the cellular tissue reaction toward collagen-based materials from different tissues. Furthermore, it became obvious that the presence of multinucleated giant cells was associated with the material breakdown/degradation and vascularization. Further clinical data are necessary to assess extent to which the presence of multinucleated giant cells observed here will influence the materials stability, integration, and, correspondingly, tissue regeneration within human tissue.
Scanning electron microscopy images of the analyzed BEGO Collagen Fleece. (a) Profile of the fleece (×20, scale bar = 1 mm). (b) and (c) The dense side of the collagen fleece. (d) and (e) The porous membrane side. (b and d: ×50, scale bars = 500 μm; c and e: ×100, scale bars = 200 μm).
Scanning electron microscopy images of the analyzed BEGO Collagen Membrane. (a) Profile of the membrane (×200, scale bar = 100 μm. (b) and (c) The dense side of the collagen membrane. (d) and (e) The porous membrane side. (b and d: ×20, scale bars = 1 mm; c and e: ×200, scale bars = 100 μm).
Integrity of the collagen fleece and results of the histomorphometric thickness measurements. (a1 through a4) The BEGO collagen fleece (CFM = double arrows) profile during the observation period (hematoxylin and eosin stainings, ×200, scale bars = 100 μm). Note that at day 60, fragmentation of the fleece was observed, so that no histologic results of this study time point were incorporated. (b) Results of the histomorphometric analysis of fleece thickness from day 3 until day 30 after implantation (* = statistically significant).
Figure 4. Representative pictures of the tissue reaction to the BEGO Collagen Fleece (CFM) within the subcutaneous connective tissue (CT). (a) Tissue reaction at day 3 after implantation. Single mononuclear cells (arrow heads) were adherent on both sides of the fleece (CFM), while a few cells have started to penetrate the fleece body (arrows) (hematoxylin and eosin [H&E] staining, ×400, scale bar = 10 μm). (b) Tissue reaction at day 10 after implantation. A compact cell-rich layer of macrophages (arrow heads) was located at the material-tissue interface; also at this time point, a few mononuclear cells (arrows) were detectable within the membrane body (F4/80 immunostaining, ×400, scale bar = 10 μm). (c) Tissue reaction at day 15 after implantation. At this time point, a fiber-rich layer was located at the material-tissue interface that contained mostly mononuclear cells (black arrow heads), but also a few multinucleated giant cells (red arrow heads). Furthermore, a higher amount of mononucleated cells (arrows) was detectable within the fleece body. Additionally, the gaps between the single collagen fibers of the fleece were now filled by fibers of the murine connective tissue (asterisk) as a sign of materials integration (Movat's pentachrome staining, ×400, scale bar = 10 μm). (d) Tissue reaction at day 30 after implantation. At this time point, the fleece showed signs of integrity reduction, which was expressed by allowing the invasion of connective tissue cords (yellow asterisks). Mono- and multinucleated giant cells (arrows/red arrow heads) infiltrated the fleece (Azan staining, ×400, scale bar = 10 μm). (d) Multinucleated giant cells (red arrow heads) in close location to microvessels (green arrows) at the peripheral region of the collagen fleece at day 30 after implantation (mononuclear cells = black arrows) (CD31 immunostaining, ×400, scale bar = 10 μm). (f) Tissue reaction at day 60 after implantation. At this time point, only small fragments of the collagen fleece were detectable within the subcutaneous connective tissue (CT). Only mononuclear cells (black arrows) and single small vessels (green arrows) infiltrated these islands (H&E staining, ×400, scale bar = 10 μm). Figure 5. Representative histologic pictures of the histochemical tartrate resistance acid phosphatase (TRAP) detection of both analyzed collagen-based materials at day 15 after implantation (a: BEGO Collagen Fleece [CFM], (b) BEGO Collagen Membrane [CM]). In both cases, the multinucleated giant cells (red arrow heads) and the mononuclear cells at the surface of the materials (black arrow heads) and within materials bodies (arrows) showed almost no signs of TRAP expression (TRAP stainings, ×400, scale bars = 10 μm).
Membrane integrity and histomorphometric analysis of membranes thickness. (a1 through a5) Collagen membrane (CM = double arrows) profile during the observation period (hematoxylin and eosin stainings, ×200, scale bars = 100 μm). (b) The histmorphometric analysis of membrane thickness from day 3 until day 60 after implantation (* = statistically significant).
Representative pictures of the multistratified collagen membrane (CM) within the subcutaneous connective tissue (CT). The left row (a1 through a5) shows the tissue reaction toward the compact surface layer of the membrane, while the right (b1 through b5) row displays the reactions to its spongy and rough surface layer. (a1) and (b1) Tissue reactions at day 3 after implantation. A multilayered wall (arrow heads) consisting of mononuclear cells was located on the surface of the compact surface layer of the collagen membrane (a1), while only single mononuclear cells (arrows) were adherent on the spongy surface of the membrane at this early study time point (b1). Only at the membrane side with the compact layer were single mononuclear cells (arrows) detectable within the membrane body (a1) (Masson Goldner-stainings, ×400, scale bars = 10 μm). (a2) and (b2) Tissue reactions to the collagen membrane at day 10 after implantation. At this time point, a cell-rich peri-implant CT was detectable at the compact surface layer of the membrane. Mononuclear cells, such as macrophages (black arrow heads) and single multinucleated cells (red arrow head), were located at the material's surface. At this time point, mononuclear cellular penetration (arrows) seemed to occur from mononuclear cells toward the inner region of the compact layer (a2). A wall of mononuclear cells (arrow heads) covered the spongy surface layer of the membrane (b2) (a2: F4/80 staining, b2: Sirius staining, ×400, scale bars = 10 μm). (a3) and (b3) Tissue reaction to the collagen membrane at day 15 after implantation. At this time point, the compact layer was covered by a high amount of multinucleated giant cells (red arrow heads) in combination with mononuclear cells (black arrow heads). Mainly single mononuclear cells (black arrows) infiltrated this membrane component (a3). In contrast, the spongy layer was covered mainly by mononuclear cells (black arrow heads), while only single multinucleated cells were detectable (red arrow head). Only single mononuclear cells (arrows) were located within the spongy membrane body at this time point (b3) (a3: Azan staining, b3: Movat's pentachrome staining, ×400, scale bars = 10 μm). (a4) und (b4) Tissue reactions to the collagen membrane at day 30 after implantation. The compact layer was completely interspersed by mononuclear (black arrow heads) and multinucleated giant cells (green arrow heads) (a4). Furthermore, the spongy layer was covered mainly by mononuclear cells, while only few of these cells (arrows) had started to infiltrate the membrane body at this time point (b4). (a4: Sirius staining, b4: Azan staining, ×400, scale bars = 10 μm). (a5) and (b5) Tissue reactions to the collagen membrane at day 60 after implantation. Only a thin remnant of the compact layer (asterisk) seemed to be remaining at this late study time point. No signs of the previously observed multinucleated tissue reaction were detectable toward this membrane part, and this membrane side was covered by mononuclear cells (arrow heads). Few cells were detected penetrating into the membrane through the compact layer (arrows) (a5). The spongy layer of the membrane was furthermore mainly covered by mononuclear cells (arrow heads), and a slight tissue ingrowth was observable. Few mononuclear cells (arrows) were located within the membrane body (b5) (a5: Sirius staining, b5: Azan staining, ×400, scale bars = 10 μm).
Results of the histomorphometric analyses. (a) Analysis results of the material-adherent multinucleated giant cells. (b) Analysis results of the vessel density (vessels/mm2). (c) Analysis results of the percent vascularization (•/••/••• = intraindividual significances, */**/*** = interindividaul significances).
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