Induction of multinucleated giant cells in response to small sized bovine bone substitute (Bio-Oss™) results in an enhanced early implantation bed vascularization

Abstract

Purpose: The host tissue reaction to the xenogeneic bone substitute Bio-Oss™ (Geistlich Biomaterials, Wolhousen, Switzerland) was investigated focusing on the participating inflammatory cells and implantation bed vascularization.

Materials and methods: Bio-Oss™ was implanted subcutaneously into CD1 mice for up to 60 days and analyzed by means of specialized histological and histomorphometrical techniques after explantation.

Results: Bio-Oss™ induced within the first 15 days an early high vascularization combined with a marked presence of multinucleated giant cells. The latter cells were associated mainly with the smaller sized granules within the implantation bed. Toward the end of the study the number of multinucleated giant cells decreased while the tissue reaction to the larger granules was mainly mononuclear.

Conclusion: The results of the present study showed that smaller xenogeneic bone substitute granules induce multinucleated giant cells, whereas the larger-sized ones became integrated within the implantation bed by means of a mononuclear cell-triggered granulation tissue. Obviously, the presence of multinucleated giant cells within biomaterial implantation beds is not only related to the type of synthetic bone substitute material, but also to the granule size of the natural-based xenogeneic bone substitute material.

Keywords: Bio-Oss; multinucleated giant cells; vascularization; xenogeneic bone substitute.

Figure 1

The tissue reaction to the xenogeneic bone substitute Bio-Oss™ at day 3 after implantation. The peri-implant tissue (CT) of the granules (BO) was characterized by a high number of vessels (a, black arrows) and mononuclear cells, that interact with material's surfaces within the periphery of the implantation bed (a and b, blue arrows). Within the interspaces of the granules a matrix composed of connective tissue fibers and fibrin can be observed (a and b, black asterisks) (Movat Pentachromestainings, a: ×200, scale bar = 100 μm, b: ×400, scale bar = 10 μm)

Figure 2

The results of the material characteristics of the xenogeneic bone substitute Bio-Oss™ (BO). (a) The bone substitute shows trabecular architecture without any signs of a lamellar substructure, while the bone matrix appeared crystalline and inhomogeneous. Furthermore, no signs of any other tissue components were identifiable (Giemsa-staining, ×100, scale bar = 100 μm). (b) Within the bone matrix osteocyte lacunae (arrows) without any cells or cell remnants were found (Giemsa-staining, ×400,scale bar = 100 μm)

Figure 3

The tissue reaction to the xenogeneic bone substitute Bio-Oss™ at day 10 after implantation. (a) The bigger Bio-Oss™ (BO) granules within the center of the implantation bed were surroundedby a vessel-(black arrows) and fiber-rich connective tissue (CT). At the granule's surfaces mainly mononuclear cells (blue arrows) beside some scattered multinucleated giant cells (blue arrow heads) were observable (Azan-staining, ×100, scale bar = 100 μm). (b) In contrast to the bigger granules (BO), which induced a tissue reaction with mainly mononuclear cells (blue arrows), the smaller granules (green asterisks) within the peripheral regions of the implantation bed were mainly surrounded by high numbers of multinucleated giant cells (blue arrow heads) and integrated within an vessel-rich (black arrows) connective tissue (H and E, ×200, scale bar = 10 μm). (c) Tartrate-resistant acid phosphatase (TRAP)-staining showed that the highest number of mononuclear cells adherent to the larger material granules (BO) were TRAP-negative (blue arrows), while only a few of these cells showed TRAP-expression (red arrows). Moreover,some of the multinucleated giant cells (red arrow heads) did express this molecule, while most of these cells were TRAP-negative (blue arrow heads) (TRAP-staining, ×400, scale bar = 10 μm). (d) The majority of the multinucleated giant cells (red arrow heads) that surrounded the smaller bone substitute granules (green asterisks) showed TRAP-expression aswell as many of the mononuclear cells (red arrows) (TRAP-staining, ×400, scale bar = 10 μm)

Figure 4

The tissue reaction to the xenogeneic bone substitute Bio-Oss™ at day 15 after implantation. (a) Also, at this time point, the bigger-sized granules (BO) were surrounded by a cell-rich connective tissue (CT), which contained numerous vessels (black arrows). Mainly mononuclear cells (blue arrows) in addition to some single multinucleated giant cells (blue arrow heads) were seen at the surface of the granules (Azan-staining, ×100, scale bar = 100 μm). (b) The small granules of Bio-Oss™ (green asterisks) at this study time point were also embedded within a vessel-(black arrows) and cell-rich connective tissue (CT) and at their surfaces the multinucleated giant cells (blue arrow heads) were still dominant (Movat Pentachrome-staining, ×200, scale bar = 10 μm). (c) The bigger granules were still surrounded byhigh numbers of tartrate-resistant acid phosphatase (TRAP)-negative mononuclear cells (blue arrows), while only a low amount of these cells were TRAP-positive (red arrows). The majority of the giant cells also showed no signs of TRAP-expression (blue arrow heads) and only single TRAP-positive multinuclear cells were present (red arrow head) (CT = connective tissue) (TRAP-staining, ×200, scale bar = 10 μm). (d) The analysis showed again that most of the giant cells that were adjacent to the smaller material granules (green asterisks) were TRAPpositive (red arrow heads). In addition, TRAP-positive (red arrows) and TRAP-negative mono-nuclear cells (blue arrows) were observed within the implantation bed (TRAP-staining, ×400, scale bar = 10 μm)

Figure 5

The tissue reaction to the xenogeneic bone substitute Bio-OssTM at day 30 after implantation. (a and b) The Bio-OssTM granules (BO) were still embedded within a fiber-rich connective tissue (CT) associated with numerous blood vessels (black arrows). Mainly mononuclear cells (blue arrows) and also a few giant cells (blue arrow heads) were still observable at the material surfaces at this time point (H and E, a: ×100, scale bar = 100 μm; b: ×200, scale bar = 10 μm). (c and d) The mononuclear cells were mainly tartrate-resistant acid phosphatase (TRAP)-negative (blue arrows) and only single cells were identifiable expressing the TRAP-molecule (black arrows). At this time point, the multinuclear cells also show mainly TRAP-positivity (red arrow heads) (TRAP-negative giant cells = black arrow heads) (BO = Bio-Oss granules) (TRAP-staining, c: ×100; d: ×400, scale bars = 10 μm)

Figure 6

The tissue reaction to the xenogeneic bone substitute Bio-OssTM at day 60 after implantation. (a and b) The intergranular connective tissue (CT) of the Bio-OssTM granules (BO) still showed a fiber-and vessel-rich (black arrows) composition. Also, at this time point most of the adherent cells were mononuclear (blue arrows) and only a minority of the reacting cells was multinuclear (blue arrow heads) (a: Masson Goldner-staining, ×100, scale bar = 100 μm; b: Azan-staining, ×400, scale bar = 10 μm). (c and d) The single multinucleated giant cells mainly showed tartrate-resistant acid phosphatase (TRAP)-expression (red arrow heads) at this time point, while only single TRAP-negative multinucleated cells (black arrow head) were found. The majority of the mononuclear cells were TRAP-negative (blue arrows). Only single TRAP-positive mononuclear cells (black arrow) were observable at this time point (TRAP-staining, c: ×100, ; d: ×400, scale bars = 10 μm

Figure 7

The histomorphometrical results. (a) Vessel density (vessels/mm²), (b) Percent vascularization (% area of vessels/area of implantation bed), (c) Multinucleated giant cells and their tartrate-resistant acid phosphatase (TRAP)-positive and TRAP negative subforms (cells/mm²)