2.1 Fabrication of DP
The cranium periosteum of mini-pigs was harvested using the following procedure. Briefly, 12-month-old minipigs were anesthetized by intraperitoneal injection of Zoletil (50 mg/kg, Virbac, France). Then the skin and the overlying fascia of the cranium were cut to expose the periosteum. Then the intact periosteum was carefully stripped from the bone surface using a periosteal detacher. The harvested natural periosteum (NP) was prepared for further evaluation or the decellularization process.
The decellularization process for the preparation of DP involved physical, chemical, and enzymatic methods. First a freeze-thaw process (-80℃, 24 h; 37℃, 2 h) was repeated three times. Then the obtained samples were treated with 2% Triton X-100 for 12 h followed by 0.5% sodium dodecyl sulfate (SDS) solution for 4 h and 100 U/ml DNase I for 12 h, after which it was soaked in ultrapure water for 3 days to remove the residual treatment reagents. Finally, the fabricated DP was stored in sterile phosphate-buffered saline (PBS) containing 100 U/ml penicillin and 100 μg/ml streptomycin for preservation.
Collagen scaffolds (COL scaffolds) for use as a control were prepared as previously described [22].