Osseointegration Effect of Micro-Nano Implants Loaded With Kaempferol in Osteoporotic Rats

Abstract

Objective: To investigate the effect of osseointegration of kaempferol loaded on the surface of micro-nanomorphic implants in ovariectomized rats. Methods: Titanium flakes were polished to obtain the PT group, anodized and acid-etched to obtain the NT and WNT groups, loaded with kaempferol to obtain the KNT and KWNT groups, and spin-coated on chitosan-gelatin composite film to obtain the KNT-CG and KWNT-CG groups. In vitro experiments were performed to observe the physicochemical properties of the titanium tablets in each group through scanning electron microscopy and contact angle experiments. The cytotoxicity and drug release pattern were observed using CCK-8 and drug release assays. An osteoporosis rat model was established. Pure titanium implants were divided into PT, NT, WNT, KNT-CG, and KWNT-CG groups after the same treatment and used in the in vivo experiments and then implanted in the femur of mice in each group. After 4 weeks, all samples were collected for toluidine blue staining, micro-computed tomography scanning, and bone morphometry analysis to evaluate their osteogenic properties. Results: According to scanning electron microscopy, the surface of the titanium flakes had a micro-nano morphology in the WNT group and the KNT and KWNT groups were functionally loaded with kaempferol. In CCK-8 and drug release experiments, the loaded kaempferol and gelatin composite membranes showed no significant toxic effects on cells. The drug release time in the KNT-CG and KWNT-CG groups was significantly longer than that in the KNT and KWNT groups, with the release time in the KWNT-CG group reaching 15 days. In vivo experiments micro-computed tomography and bone morphometry analysis showed that the osteoporosis model had been successfully constructed. The bone volume fraction around the implant increased. Toluidine blue staining showed new bone formation and a significantly increased number of bone trabeculae. Conclusion: Kaempferol micro-nanocomposite coating improved the osseointegration ability of implants in osteoporotic rats.

Keywords: Kaempferol; Osseointegration; Osteoporosis; chitosan; gelatin; implants; micro-nano.

FIGURE 1

Animal experimental procedures.

FIGURE 2

Surface morphology of each titanium sheet under SEM. (A): PT group; (B): Micron structure; (C): NT group; (D): WNT group; (E): KNT group; (F): KWNT group; (G): KNT-CG group; (H): KWNT-CG group.

FIGURE 3

Analysis of the results of contact angle comparison between groups. Bar graphs: Data are presented as mean ± SD. ^# represents comparison with NT group, ^## p < 0.01; * represents comparison with PT group, **p < 0.01.

FIGURE 4

Analysis of CCK-8 results.

FIGURE 5

Concentration-absorbance standard curve of kaempferol solution.

FIGURE 6

Cumulative release curve of titanium tablets loaded with kaempferol.

FIGURE 7

Micro-CT scan section of rat femur. Compared to the sham operation (SHAM) group, the bilateral ovariectomized (OVX) group had reduced bone trabecular density and successful osteoporosis modeling.

FIGURE 8

3D Micro-CT scan of rat femur. (A):PT group; (B):NT group; (C): WNT group; (D):KNT-CG group; (E):KWNT-CG group.

FIGURE 9

Morphometric analysis of rat femur. BV/TV, bone volume per tissue volume; DA, degree of anisotropy; Tb.N, trabecular number; Tb.Th, trabecular thickness; Tb. Sp, trabecular sepacing. Bar graphs: Data are presented as mean ± SD. ^# represents comparison with NT group, ^## p < 0.01, ^# p < 0.05; *represents comparison with PT group,**p < 0.01; *p < 0.05.

FIGURE 10

Rat femur toluidine blue staining, dark blue part represents new bone tissue. (A): PT group; (B): NT group; (C): WNT group; (D): KNT-CG group; (E): KWNT-CG group.