3 Results
3.1 Effectiveness ofthe decellularization procedure
Macroscopic images of NP and DP are shown in Figure 1A. HE staining of both sample types showed a double-layered (fibrous layer and cambium layer) structure consistent with NP as well as well-organized collagen fibers, but cell nuclei were absent from both layers of DP. Within DP, collagen fibers were still arranged in an orderly manner similar to that in NP (Figure 1B). DAPI staining also confirmed that almost no cellular components or nuclear material could be observed in DP (Figure 1C). The DNA quantification analysis indicated that the amount of DNA in NP was 526.2±44.68 ng/mg, while that in DP was 38.2±12.39 ng/mg (Figure 1D). The quantification of collagen content showed no significant difference between NP and DP (115.76±11.26 and 103.20±13.26 μg/mg dry weight for NP and DP, respectively, P>0.05; Figure 1E). The DMMB results indicated a significant decrease in GAG content in DP (1.87±0.07 μg/mg dry weight) compared with NP (3.99±0.07 μg/mg dry weight, P<0.05; Figure 1F). In addition, only examination of DP and not NP showed no visible bands of DNA detected by agarose gel electrophoresis. In summary, these results indicate the effectiveness of the decellularization procedure for removal of cellular components from periosteum.