Fig 1.
The morphologies of xenograft bones deproteinized by different methods.
(A) The macroscopic view of H2O2 and pepsin deproteinized bovine cancellous bones. (B) The microscopic view (40×: upper panel, 200×: bottom panel) of Ctl, H2O2 and pepsin deproteinized bovine cancellous bones. The deproteinized bones were decalcified prior to paraffin section and HE staining, the arrow indicates adipose tissue. (C) SEM images of Ctl, H2O2 and pepsin deproteinized bovine cancellous bones. Ctl: non-deproteinized fresh bones.
Table 1.
The mean value of pore size and porosity in each groups.
Fig 2.
The biochemical and biomechanical analysis of H2O2 and pepsin deproteinized xenograft bones (n≥5) (A) The protein content (g/100g) and hydroxyproline content (μg/mg) of bovine cancellous bones in different groups. Bones were ground and subjected to the Kjeldahl method and HPLC-MS for protein content and hydroxyproline content, respectively. (B) The biomechanical properties of each group. No differences were observed in maximum compression load (Mpa) and bending load (Mpa).
Compared with Ctl, *P < 0.05. Compared with H2O2, #P < 0.05.
Fig 3.
(A) In vitro cellular affinity assay showed high cell proliferation of osteoblasts in pepsin treated bones (left) and H2O2 treated bones (right) (magnification × 550). (B) The AKP activity was significantly increased in osteoblasts and pepsin treated bones co-culture system.
Compared with Ctl, ***P < 0.001. Compared with H2O2, ###P < 0.001.
Fig 4.
Quantification of calcium deposits of osteoblast cultured on pepsin treated bones, H2O2 treated bones and non-deproteinized fresh bones on day 14 (left) and day 28 (right).
Compared with Ctl, *P < 0.05. Compared with H2O2, #P < 0.05.
Fig 5.
The X-ray analysis showed the osteogensis performance of different graft materials (n = 5).
Different graft materials were implanted in defect sites, the X-ray images were taken 0 day (D 0), 4 weeks (4 w), 8 weeks (8 w) and 12 weeks (12 w) after bone grafting, respectively. The X-ray results were scored by Lane-Sandhu grading method and the statistical data were shown in the right panel. Compared with H2O2, *P < 0.05.
Fig 6.
The micro-CT analysis showed the bone quantity and quality in different groups (n = 5).
The following parameters were measured at the indicated time points: bone volume fraction (BVF), tissue mineral content (TMC), tissue mineral density (TMD), trabecular number (Tb.N), trabecular separation (Tb.sp), trabecular thickness (Tb.th) and structure model index (SMI). Compared with H2O2, *P < 0.05.
Fig 7.
The histological analysis of bones in the defect sites of different groups.
The defect sites of radius in each group were dissected and subjected to HE staining. The pictures on the left side of each group represent the low power microscopic views (40 ×); the magnified views of boxed areas (200 ×) were shown on the right side of each group.
Fig 8.
Pepsin deproteinized xenograft bones reduces immune-reactivity upon bone grafting.
The activity of cellular immunity (CD4+/CD8+ ratio) was significantly higher in H2O2 group when compared with that in the pepsin group (A); humoral immunity activity (IgG level) was expressed at a lower level at the donor site in the pepsin group (B).