Evaluation of the osteogenesis and osseointegration of titanium alloys coated with graphene: an in vivo study

Abstract

The aim of this study was to investigate whether a surface coating with graphene could enhance the surface bioactivation of titanium alloys (Ti₆Al₄V) to further accelerate in vivo osteogenesis and osseointegration at the implant surface. In this study, a New Zealand white rabbit femoral condyle defect model was established. After 4, 12 and 24 weeks, biomechanical testing, micro-computed tomography (Micro-CT) analyses and histological observations were performed. At the highest push-out forces during the test, microstructure parameters, such as the bone volume/total volume fraction (BV/TV) and mineral apposition rate (MAR), of the new bone were significantly higher in the graphene-coated Ti₆Al₄V group (G-Ti₆Al₄V) than in the Ti₆Al₄V group (P < 0.05). Van Gieson (VG) staining showed that the G-Ti₆Al₄V group had more new bone formation than the Ti₆Al₄V group, and the G-Ti₆Al₄V group showed a closer fit between the bone and implant. In conclusion, graphene might be a novel type of nano-coating material for enhancing the surface biological activity of Ti-based alloy materials and may further promote in vivo osteogenesis and osseointegration.

Figure 1

(a) Schematic diagram of the processes used for the surface etching and transfer of graphene to a copper substrate and the modification of the scaffold. (b) G-Cu: the entire surface is covered with a layer of gray graphene film (n = 5, graphene coverage of 100%). (c) SEM Ti₆Al₄V image: only scratches ae visible on the surface after polishing (n = 5). (d) G-Ti₆Al₄V: the black arrows point to the graphene film and wrinkles (n = 5). (e) G-Ti₆Al₄V: after 1 h of sonication, the graphene coating (black arrows) was still stable (n = 5). (f) Raman spectroscopy (laser wavelength = 630 nm): the nattier blue peak corresponds to G-Ti₆Al₄V after 1 h of sonication, the red peak corresponds to G-Ti₆Al₄V, the deep blue peak corresponds to G-Cu, and the characteristic graphene G peak (1,580 cm⁻¹) and 2D peak (2,660 cm⁻¹) are clearly present. The green peak corresponds to the uncoated Ti₆Al₄V, which does not show the two characteristic graphene peaks in the Raman spectra (n = 5).

Figure 2

(a) Completely removed femoral condyle. (b,c) After trimming, the condyle was placed into a special fixture. (d,e,f) Push-out test: the load speed was set to 1 mm/min, and the maximum failure load was recorded. (g) Maximum failure load of the two groups after 4, 12 and 24 weeks (n = 6). The results are expressed as the mean ± SD; *represents a significant difference (P < 0.05).

Figure 3

(a) Implanted scaffolds and new bone after 4, 12, and 24 weeks; the establishment of a unified region of interest and subsequent image reconstruction were performed through Micro-CT; yellow represents new bone, and white represents the implant. Scale: 1.5 mm. (b) Bone volume fraction (BV/TV) of the two groups obtained from analysis of the Micro-CT data (n = 6). (c) Trabecular number (TbN) and (d) trabecular spacing (Tb.Sp) (n = 6). The results are expressed as the mean ± SD; *represents a significant difference (P < 0.05).

Figure 4

(a) Double-labeling immunofluorescence images of the G-Ti₆Al₄V group and Ti₆Al₄V group (Sections in the center of four scaffolds) under a fluorescence microscope. The top four images are coated titanium alloys (G-Ti6Al4V), and the bottom four are blank uncoated Ti6Al4V. After blue light irradiation, under microscopy, calcein showed green, and tetracycline showed yellow. Scale: 100 µm. (b) Mineral apposition rate of bone in the two groups (n = 6). The results are expressed as the mean ± SD; *represents a significant difference (P < 0.05).

Figure 5

(a) Histological images of VG-stained hard tissue sections at 4, 12 and 24 weeks (n = 6). The implanted material (Implant) is stained black. New bone tissue (B) is stained red. Fibrous tissue (F) is stained blue. Scale: 100 µm. (b) New bone volume fraction/total bone volume (BV/TV) of the two groups was analyzed using Image-Pro Plus 6 software (n = 6).

Figure 6

(a) Material specifications. (b–e) Demonstration of the surgical procedure. (g) X-ray images after the operation.