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].