Surgery

Rats were anesthetized by ketamine 100 mg kg⁻¹ i.m. and xylazine hydrochloride 5 mg kg⁻¹ i.m., and a 5-mm standardized critical size calvarial defect was created, as previously described [15]. In short, a circular bone disk was removed from the left parietal bone using a trephine drill with an outer diameter of 5 mm. The created ≈ 20 mm² critical size defect was covered with a commercially available, bilayer pure collagen type I and III membrane placed either regularly or upside-down, in accordance with the randomized treatment allocations. The membrane overlapped the defect perimeter by at least 1 mm at every point. The membrane was not fixed to the bone. Wounds were closed in two layers with resorbable USP 5–0 sutures. Butorphanol 1.25 mg kg⁻¹ s.c. and meloxicam 0.15 mg kg⁻¹ s.c. were used to control postoperative pain. Rats were sacrificed after 4 weeks of healing by an intracardial overdose of thiopental.