Degradation, Bone Regeneration and Tissue Response of an Innovative Volume Stable Magnesium-Supported GBR/GTR Barrier Membrane

Abstract

Introduction: Bioresorbable collagenous barrier membranes are used to prevent premature soft tissue ingrowth and to allow bone regeneration. For volume stable indications, only non-absorbable synthetic materials are available. This study investigates a new bioresorbable hydrofluoric acid (HF)-treated magnesium (Mg) mesh in a native collagen membrane for volume stable situations.

Materials and methods: HF-treated and untreated Mg were compared in direct and indirect cytocompatibility assays. In vivo, 18 New Zealand White Rabbits received each four 8 mm calvarial defects and were divided into four groups: (a) HF-treated Mg mesh/collagen membrane, (b) untreated Mg mesh/collagen membrane (c) collagen membrane and (d) sham operation. After 6, 12 and 18 weeks, Mg degradation and bone regeneration was measured using radiological and histological methods.

Results: In vitro, HF-treated Mg showed higher cytocompatibility. Histopathologically, HF-Mg prevented gas cavities and was degraded by mononuclear cells via phagocytosis up to 12 weeks. Untreated Mg showed partially significant more gas cavities and a fibrous tissue reaction. Bone regeneration was not significantly different between all groups.

Discussion and conclusions: HF-Mg meshes embedded in native collagen membranes represent a volume stable and biocompatible alternative to the non-absorbable synthetic materials. HF-Mg shows less corrosion and is degraded by phagocytosis. However, the application of membranes did not result in higher bone regeneration.

Keywords: GBR/GTR membrane; barrier membrane; collagen; degradation; histomorphometry; in vivo; magnesium; tissue reaction.

Figure 1

Common strategies to passivate magnesium for in vitro/in vivo application.

Figure 2

Comparison of untreated and processed samples. (A) Pictures of both an untreated and HF-treated magnesium sheet for the in vitro experiments. (B) Processed magnesium meshes before embedded into a collagen membrane.

Figure 3

Different membrane modifications. (A) Integration of the magnesium mesh into the collagen fleece. (B,C) Moldability and stability of the collagen membrane with an integrated Mg mesh.

Figure 4

Cytocompatibility results using L9292 cells of the different variants. (A) proliferation measured by a BrdU assay; (B) viability measured by a Sodium 3,3′-[1(phenylamino)carbonyl]-3,4-tetrazolium]-3is(4-methoxy-6-nitro) Benzene Sulfonic acid Hydrate (XTT)-assay; (C) cytotoxicity measured by a Lactate Dehydrogenase (LDH) assay. Values are either normalized against positive controls (LDH) or negative control (XTT, BrdU). Means with error bars indicating standard deviations. The dotted line indicates thresholds which should not be exceeded (LDH) or fall below (XTT; BrdU). Significant differences are indicated (•: p < 0.05, **: p < 0.01). (D) Both untreated and HF-treated magnesium after 72 h extraction. The untreated magnesium shows enhanced corrosions, which is visible due to the high surface porosity and black textured corrosion products. The HF-treated magnesium was not different from the initial morphology.

Figure 5

Attachment of cells on surfaces of the different variants and controls. The pictures show the attachment, vitality and morphology of the cells. Green: vital cells; red: dead cells. Spindle-shaped morphology indicates healthy cells with firm attachment. Round cells indicate poor attachment onto the surface.

Figure 6

Histopathological comparison of both treated and HF-treated membranes. Images of Masson-Goldner (C,D) and Von Kossa (A,B,E,F) staining of the implantation site at 6, 12 and 18 weeks (100× magnifications, scalebars = 100 µm). Left for untreated (Mg) and right for HF-treated (Mg^#) magnesium. Mg is mainly degraded via dissolution and scarcely through phagocytic processes. Mg-HF however, is primarily being resorbed via active phagocytosis and non-cellular dissolution only plays a minor role. After degradation of the HF-coating, decomposition, as with untreated Mg, principally occurs non-cellular-driven but through dissolution. Yellow arrows = phagocytic cells, black arrows = fibroblasts, asterisks = slight fibrosis, white arrows = septa between the gas cavities.

Figure 7

Gas cavity formation of HF- and untreated membranes.Representative images of Masson-Goldner (A,F) and Von Kossa (B–E) staining (40×) of the implantation site (scalebar = 20 µm) at 6, 12 and 18 weeks. Left for untreated (Mg) and right for HF-treated (Mg^#) magnesium. Fibrotic capsule forming (*) is visible at all times whereas gas cavity formation ceased to show after 12 weeks. The HF-coated mesh was mainly degraded by mononuclear cells (yellow arrows) up to 12 weeks, while the uncoated magnesium meshes elicited a fibrosis-like tissue reaction showing fibroblast accumulation (black arrows). Starting from 18 weeks after implantation, the tissue reaction in both groups was similar. Yellow arrows = phagocytic cells, black arrows = fibroblasts, asterisks = slight fibrosis.

Figure 8

Overview of the various quantitative measurements. (A) Gas cavity surface in µm² for uncoated (Mg; blue) and HF-treated (Mg-HF; red) magnesium after 6 and 12 weeks. HF-treated magnesium meshes show significantly lower gas cavity development compared to untreated magnesium meshes (**: p < 0.01) up to 6 weeks after implantation. Gas cavity dimension of untreated magnesium meshes shows to be reduced significantly after 12 weeks (•: p < 0.05). (B–D) Bone regeneration as measured with contact radiography, DVT and in histomorphometry after 6, 12 and 18 weeks for uncoated (Mg; blue) and HF-coated magnesium (Mg-HF; red), collagen (Collagen; green) and control (Control; black). No significant differences (p > 0.05) were detectable.

Figure 9

Imaging procedures to detect bone regeneration. (A) Three-dimensional DVT reconstruction and (B) radiological image as used for bone surface regeneration measurements. Selected circular defect area (C) before and (D) after application of color threshold.

Figure 10

Intraoperative view on calvarial implantation model. Creation of four circular defects using a trephine burr (A,B), followed by placement of membranes (C). Image of the hydrofluoric acid (HF)-treated magnesium (Mg) mesh in a native collagen membrane (D).