Longevity of collagen in membrane applied in double-layer technique

Objective: This study histologically evaluates and compares the bio-degradation of two layers and a single layer of collagen membrane. Methods: Two 5-mm in diameter calvarial defects were surgically made in each of 24 Wistar rats. One Layer (MLM) or two layers (DLM) of Bio-Gide® membrane discs labeled with biotin were placed in the bony defects. Rats were sacrificed at 4 or 9 weeks. Decalcified sections and free labeled BG discs were stained with Horseradish peroxidase-conjugated Streptavidin for detection of biotinylated collagen using Aminoethyl Carbazole as substrate. The amount of collagen and the thickness of the membranes were measured by image analysis software. Statistical analysis consisted of ANOVA with repeated measures and pair T-tests. Results: Double-layer membrane sites presented significantly greater amount of collagen compared with single-layer sites (p<0.001) at all time points. Furthermore, the amount of residual collagen at 4 weeks was significantly greater than that at 9 weeks (p=0.005). At four weeks one-layer and two-layer membrane contained 0.047±0.034 mm² and 0.090±0.050 mm² collagen per measurement window, respectively. At 9 weeks, collagen area per window reduced to 0.025 ±0.016 mm² in mono-layer and 0.037±0.026 mm² in double layer membranes. The area of residual collagen was significantly and negatively related to time (p=0.021). Comparison of residual collagen in the MLM and DLM to baseline showed 60% and 80% collagen degradation rate in both following 4 and 9 weeks respectively. DLM were 90% thicker than MLM at 4 weeks, and 87% at 9 (p< 0.001), with 30% reduction in thickness of MLM and 33% for DLM between 4 to 9 weeks. Conclusions: DLM increases the longevity and stability of the collagen in the membrane compared with MLM and thus may improve its barrier function in the rat; this may improve the results of GBR procedures.