Systematic evaluation of three porcine-derived collagen membranes for guided bone regeneration

Abstract

Guided bone regeneration is one of the most common surgical treatment modalities performed when an additional alveolar bone is required to stabilize dental implants in partially and fully edentulous patients. The addition of a barrier membrane prevents non-osteogenic tissue invasion into the bone cavity, which is key to the success of guided bone regeneration. Barrier membranes can be broadly classified as non-resorbable or resorbable. In contrast to non-resorbable membranes, resorbable barrier membranes do not require a second surgical procedure for membrane removal. Commercially available resorbable barrier membranes are either synthetically manufactured or derived from xenogeneic collagen. Although collagen barrier membranes have become increasingly popular amongst clinicians, largely due to their superior handling qualities compared to other commercially available barrier membranes, there have been no studies to date that have compared commercially available porcine-derived collagen membranes with respect to surface topography, collagen fibril structure, physical barrier property, and immunogenic composition. This study evaluated three commercially available non-crosslinked porcine-derived collagen membranes (Striate+^TM, Bio-Gide^® and Creos^TM Xenoprotect). Scanning electron microscopy revealed similar collagen fibril distribution on both the rough and smooth sides of the membranes as well as the similar diameters of collagen fibrils. However, D-periodicity of the fibrillar collagen is significantly different among the membranes, with Striate+^TM membrane having the closest D-periodicity to native collagen I. This suggests that there is less deformation of collagen during manufacturing process. All collagen membranes showed superior barrier property evidenced by blocking 0.2-16.4 μm beads passing through the membranes. To examine the immunogenic agents in these membranes, we examined the membranes for the presence of DNA and alpha-gal by immunohistochemistry. No alpha-gal or DNA was detected in any membranes. However, using a more sensitive detection method (real-time polymerase chain reaction), a relatively strong DNA signal was detected in Bio-Gide^® membrane, but not Striate+^TM and Creos^TM Xenoprotect membranes. Our study concluded that these membranes are similar but not identical, probably due to the different ages and sources of porcine tissues, as well as different manufacturing processes. We recommend further studies to understand the clinical implications of these findings.

Keywords: barrier membrane; collagen membrane; dental implant; guided bone regeneration; immunogen.

Figure 1. Scanning electron microscopy of porcine collagen membranes. (A, B) Smooth (A) and rough (B) sides of Striate+^TM, Bio-Gide^® and Creos^TM Xenoprotect. The smooth side of the collagen membranes showed a more uniform, smooth, and organized structure than the rough side. Scale bars: 100 μm (upper row), 2 μm (middle row), 100 nm (lower row). Representative scanning electron microscopy images of both smooth and rough sides of membranes at 150k× were analysed by OrientationJ for the orientation of fibres. Striate+^TM and Creos^TM Xenoprotect showed more uniform fibre orientation than Bio-Gide^® on smooth side, whereas all three membranes showed random fibre orientation on rough side. (C, D) Porosity of smooth (C) and rough (D) sides of collagen membranes. Data are presented as means ± SEM. **P < 0.01, ***P < 0.001 (one-way analysis of variance with Tukey’s post hoc multiple comparison). ns: no significance.

Figure 2. (A, B) Diameter (A) and D-periodicity (B) of collagen bundles in Striate+^TM, Bio-Gide^® and Creos^TM Xenoprotect (Creos^TM X.). Data are presented as means ± SEM. **P < 0.01, ***P < 0.001 (one-way analysis of variance with Tukey’s post hoc multiple comparison). ns: no significance.

Figure 3. Thickness of porcine collagen membranes measured by micro-computed tomography. (A) Iodine-stained Striate+^TM, Bio-Gide^® and Creos^TM Xenoprotect (Creos^TM X.) membranes were aligned in polypropylene tubes and scanned by micro-computed tomography. (B) Cross sections of Striate+^TM, Bio-Gide^® and Creos^TM Xenoprotect membranes were extracted by AVIZO software. (C) Thickness of three samples of each membrane. Data are presented as means ± SEM. At least 11 measurements were done on each membrane. **P < 0.01, ***P < 0.001 (one-way analysis of variance with Tukey’s post hoc multiple comparison). ns: no significance.

Figure 4. Barrier properties of three porcine collagen membranes measured by gravity-based filtration. (A) Representative results of collagen membranes’ barrier property by filtration with mixed standard beads in different sizes. (B, C) Quantitative analysis of beads with all small (B, 220, 450, 880, and 1250 nm) and large (C, 2.0, 3.3, 5.2, 7.88, 10.1, and 16.4 μm) sizes passing through different collagen membranes. Data are presented as means ± SEM. **P < 0.01, ***P < 0.001 (one-way analysis of variance with Tukey’s post hoc multiple comparison). FP1: Whatman Filter paper Grade 1; FP5: Whatman Filter paper Grade 5; ns: no significance.

Figure 5. Heamatoxylin and eosin staining of different porcine collagen membranes. No heamatoxylin staining was found in all membranes. Scale bars: 50 μm. Creos^TM X.: Creos^TM Xenoprotect.

Figure 6. Anti-DNA immunostaining on different porcine collagen membranes. No DNA was found in all membranes by anti-DNA immunostaining. Positive control (Ctrl) indicates porcine aortic valve. Scale bar: 100 μm. Creos^TM X.: Creos^TM Xenoprotect; dsDNA: double stranded DNA.

Figure 7. Determination of DNA content in different collagen membranes by real-time polymerase chain reaction. Among the three collagen membranes, only Bio-Gide^® membrane showed significant DNA signal content. Data plotted above red dot line indicates undetectable within 40 cycles. Creos^TM X.: Creos^TM Xenoprotect; Ct: cycle threshold.

Figure 8. α-gal expression in different collagen membranes. No α-gal signal was detected in Creos^TM Xenoprotect, Striate+^TM and Bio-Gide^® by anti-α-gal immunostaining. Positive control (Ctrl) indicates porcine aortic valve. Scale bar: 100 μm. α-gal: alpha-gal, galactose-α-1,3-galactose; Creos^TM X.: Creos^TM Xenoprotect.