Fig 1.
Advaned bone analysis (ABA) by Micro View software.
After micro-CT image reconstruction, Micro View could analyze the BMD and microstructure of any certain volume of ROI within the femoral heads.
Fig 2.
SEM images of internal fixation screws.
The surface of nanographene coated screws (right) is covered with a thin layer of laminated material, and the morphology of the surface had clearly not been changed, as shown in the scanning electron microscopy (x1000).
Fig 3.
Osteoblast cytotoxicity test of nanographene coated screws.
The result of the cytotoxicity test of nanographene coated screws showed that nanographene coating did not affect the biocompatibility of screws, because there was no significant difference between the control group and experimental groups.
Fig 4.
HUVECs cytotoxicity test of nanographene coated screws.
The result of HUVECs cytotoxicity test showed that there was no significant difference between the control group and experimental groups as well.
Fig 5.
The curve (C) showed that the sustained release of VEGF on nanographene coating could last 9 days without a burst release. As the control gourp (B), nanographene coating screws without VEGF loaded had no VEGF released. There had very little VEGF burst relese on screws without nanographene coating (A) had no sustainded release effect.
Table 1.
The amount of sustained-release VEGF in three different groups(n = 3).
(±s).
Fig 6.
The biological activity of released VEGF.
The cumulative sustained release of VEGF within 9 days still had biological activity and could promote the tube formation of HUVECs (right) compared with the control groups (left) in vitro.
Fig 7.
The total length of tube formation of HUVECs.
The total length of tube formation in both the experimental groups and control groups were analyzed by Image-Pro Plus 6.0 software. The figure shows that the total length of tube formation in the experimental groups was higher than the control groups (P<0.05).
Fig 8.
After surgery (day 1), the fractures in both the experimental group (a) and control group (b) were clear and received good reduction. By 8 weeks postoperatively, the fracture line disappeared in the experimental groups (c), and BMD in the fracture site was similar with surrounding bone tissue. In the control group (d), the fracture line was blurred and BMD was lower than the bone tissue around the fracture site. At 12 weeks after surgery, the fracture lines in both groups disappeared. The BMD of the fracture site in the experimental group (e) was consistent with the surrounding bone tissue; however, the BMD in the control group (f) was still lower than the neighboring bone tissue.
Fig 9.
The MIP of microangiography of the femoral heads.
The MIP of microangiography of the femoral heads shows that the number and branches of microvessels in the experimental group (VEGF) was higher than the control group (CON).
Fig 10.
3D images of microvessels and bone structure.
The three-dimensional images of microvessels and bone structure (left) in the femoral heads could be displayed simultaneously through Isosurface of Micro View software. By choosing the appropriate CT values, the information of microvessels (right) could be extracted separately. The images demonstrated that the microvessels in the experimental group (a,b) were more evenly and broadly distributed than in the control group (c,d).
Table 2.
The BMD and Tb.Th of the femoral heads after micro-CT analysis at 12 weeks postoperatively (n = 5).
Fig 11.
Histopathological evaluation of the femoral heads.
HE staining (x100), Masson’s trichrome staining (x100), and CD31 immunohistochemical staining (x200) of the femoral heads at 12 weeks after surgery. a, c, e experimental groups; b, d, f control groups. a, b, c, d show the bone tissue adjacent to the screw trajectory.
Table 3.
Data analysis of trabecular bone of HE staining (n = 5).
(±s).
Table 4.
Data analysis of new bone formation area (%) of Masson’s trichrome staining (n = 5).
(±s).
Table 5.
Microvessel density of CD31 immunohistochemical staining (n = 5).
(±s).