BackgroundBilayer collagen membranes are routinely used in guided bone/tissue regeneration to serve as osteoconductive scaffolds and prevent the invasion of soft tissues. It is recommended to place the membranes with their dense layer towards the soft tissue and their porous layer towards the bony defect area. However, evidence supporting this recommendation is lacking. This study aimed to determine whether the alignment of bilayer collagen membranes has an effect on bone regeneration.MethodsIn two groups of ten male Sprague-Dawley rats each, a 5-mm calvarial defect was created. Thereafter, the defect was randomly covered with a bilayer, resorbable, pure type I and III collagen membrane placed either regularly or upside-down (i.e., dense layer towards bone defect). After 4 weeks of healing, micro-computed tomography (μCT), histology, and histomorphometry of the inner cylindrical region of interest (4.5 mm in diameter) were performed to assess new bone formation and the consolidation of the collagen membrane in the defect area.ResultsQuantitative μCT showed similar bone volume (median 8.0 mm3, interquartile range 7.0–10.0 vs. 6.2 mm3, 4.3–9.4, p = 0.06) and trabecular thickness (0.21 mm, 0.19–0.23 vs. 0.18 mm, 0.17–0.20, p = 0.03) between upside-down and regular placement, both leading to an almost complete bony coverage. Histomorphometry showed comparable new bone areas between the upside-down and regularly placed membranes, 3.9 mm2 (2.7–5.4) vs. 3.8 mm2 (2.2–4.0, p = 0.31), respectively. Both treatment groups revealed the same regeneration patterns and spatial distribution of bone with and without collagen fibers, as well as residual collagen fibers.ConclusionsOur data support the osteoconductive properties of collagen membranes and suggest that bone regeneration is facilitated regardless of membrane layer alignment.