In vivo Implantation of a Bovine-Derived Collagen Membrane Leads to Changes in the Physiological Cellular Pattern of Wound Healing by the Induction of Multinucleated Giant Cells: An Adverse Reaction?

Abstract

The present study evaluated the tissue response toward a resorbable collagen membrane derived from bovine achilles tendon (test group) in comparison to physiological wound healing (control group). After subcutaneous implantation in Wistar rats over 30 days, histochemical and immunohistochemical methods elucidated the cellular inflammatory response, vascularization pattern, membrane protein and cell absorbance capacity. After 30 days, the test-group induced two different inflammatory patterns. On the membrane surface, multinucleated giant cells (MNGCs) were formed after the accumulation of CD-68-positive cells (macrophages), whereas only mononuclear cells (MNCs) were found within the membrane central region. Peri-implant vascularization was significantly enhanced after the formation of MNGCs. No vessels were found within the central region of the membrane. Physiological wound healing revealed no MNGCs at any time point. These dynamic changes in the cellular reaction and vascularization within the test-group are related typical indications of a foreign body reaction. Due to the membrane-specific porosity, mononuclear cells migrated into the central region, and the membrane maintained its integrity over 30 days by showing no breakdown or disintegration. The ex vivo investigation analyzed the interaction between the membrane and a blood concentrate system, liquid platelet-rich fibrin (liquid PRF), derived from human peripheral blood and consisting of platelets, leukocytes and fibrin. PRF penetrated the membrane after just 15 min. The data question the role of biomaterial-induced MNGCs as a pathological reaction and whether this is acceptable to trigger vascularization or should be considered as an adverse reaction. Therefore, further pre-clinical and clinical studies are needed to identify the types of MNGCs that are induced by clinically approved biomaterials.

Keywords: adverse reaction; collagen-based biomaterial; disintegration; integration; memebrane; multinucleated giant cells; regeneration; wound healing.

Figure 1

Ex vivo interaction between liquid platelet-rich fibrin and the collagen membrane SB. (A) A control of the SB illustrating the membrane-specific porous structure (H and E staining; x10 magnification; scale bar = 100 μm). (B) Total penetration of leukocytes and platelets from liquid PRF into the SB central region (H and E staining; x100 magnification; scale bar = 100 μm). (C) High magnification micrograph showing the fibrin network (^*) within the SB collagen fibers (H and E staining; x400 magnification; scale bar = 20 μm). (D) High magnification micrograph showing the leukocytes (black arrows) within the SB collagen fibers (H and E staining; x200 magnification; scale bar = 100 μm). (E) High magnification micrograph showing the platelets (black arrows) and fibrin network (^*) within the SB collagen fibers (anti CD-61 staining; x400 magnification; scale bar = 20 μm).

Figure 2

The collagen membrane SB within the implantation bed over the investigates time points. (A) after 3 days; (B) after 10 days; (C) after 15 days; (D) after 30 days. (Azan staining; x40 magnification; scale bars = 500 μm). (E,F) Histomorphometrical image of the membrane thickness and percentage thickness over 30 days (^*p < 0.05; ^**p < 0.01; ^***p < 0.001; and ^****p < 0.0001).

Figure 3

(A) Cellular and connective tissue infiltration (black arrows) of the collagen membrane (SB) on day 15, (Masson Goldner staining; x100 magnification; scale bar = 100 μm). (B) TRAP- negative MNGCs (black arrows) on the membrane surface (SB) on day 15, (TRAP staining; x400 magnification; scale bar = 20 μm). (C) Cellular and connective tissue infiltration (black arrows) of the collagen membrane (SB) on day 30, (Masson Goldner staining; x100 magnification; scale bar = 100 μm). (D) TRAP-negative MNGCs (black arrows) on the membrane surface (SB) on day 30, (TRAP staining; x400 magnification; scale bar = 20 μm).

Figure 4

The behavior of macrophage (black arrows) accumulation on days 10 (A), 15 (B), and 30 (C) on the surface of the biomaterial (CD-68 immunohistochemical staining; x200 magnification; scale bar = 100 μm).

Figure 5

Histomorphometrical analysis. (A) The numbers of CD-68-positive cells (macrophages) per square millimeter over the time. (B) The vascularization pattern over the time in vessels per square millimeter in comparison to the control group. (C) The numbers of multinucleated giant cells (MNGCs) per square millimeter over the time. (D) The percent vascularization of the implantation bed over the time in comparison to the control group. (^*/•p < 0.05; ^**/••p < 0.01; ^{***/•••}p < 0.001; and ^{****/••••}p < 0.0001).

Figure 6

The vascularization pattern (A) on day 10 and (B) on day 15 highlighted with immunohistochemical staining of SMA in x200 magnification. Black arrow heads, vessels; scale bar, 100 μm. (C) The correlation between the induced MNGCs and the vascularization in the test group.