Evaluation of biological properties of electron beam melted Ti6Al4V implant with biomimetic coating in vitro and in vivo

Abstract

Background: High strength porous titanium implants are widely used for the reconstruction of craniofacial defects because of their similar mechanical properties to those of bone. The recent introduction of electron beam melting (EBM) technique allows a direct digitally enabled fabrication of patient specific porous titanium implants, whereas both their in vitro and in vivo biological performance need further investigation.

Methods: In the present study, we fabricated porous Ti6Al4V implants with controlled porous structure by EBM process, analyzed their mechanical properties, and conducted the surface modification with biomimetic approach. The bioactivities of EBM porous titanium in vitro and in vivo were evaluated between implants with and without biomimetic apatite coating.

Results: The physical property of the porous implants, containing the compressive strength being 163 - 286 MPa and the Young's modulus being 14.5-38.5 GPa, is similar to cortical bone. The in vitro culture of osteoblasts on the porous Ti6Al4V implants has shown a favorable circumstance for cell attachment and proliferation as well as cell morphology and spreading, which were comparable with the implants coating with bone-like apatite. In vivo, histological analysis has obtained a rapid ingrowth of bone tissue from calvarial margins toward the center of bone defect in 12 weeks. We observed similar increasing rate of bone ingrowth and percentage of bone formation within coated and uncoated implants, all of which achieved a successful bridging of the defect in 12 weeks after the implantation.

Conclusions: This study demonstrated that the EBM porous Ti6Al4V implant not only reduced the stress-shielding but also exerted appropriate osteoconductive properties, as well as the apatite coated group. The results opened up the possibility of using purely porous titanium alloy scaffolds to reconstruct specific bone defects in the maxillofacial and orthopedic fields.

Figure 1. The flow diagram showed the design of electron beam melting (EBM) porous Ti6Al4V implant with CAD for repair of mandibular bone defect:

(1) acquisition of the CT data of the patients; (2) design with CAD and fabrication of custom EBM porous titanium implant; (3) implantation of the patient specific porous implant; (4) reconstruction of the bone defect.

Figure 2. Characterization of porous Ti6Al4V samples.

(A) Porous Ti6Al4V implants fabricated by electron beam melting process. (B) Reconstructed 3D micro-CT image of the porous implant with honey-like structure. SEM images of porous Ti6Al4V samples with (C) honeycomb-like structure, (D) orthogonal structure, (E) layer structure.

Figure 3. Digital topographic images of the sample surface.

The images exhibited a rough anisotropic surface with the roughness R_a values of the (A) top and (B) lateral surfaces being in the range of 5–10 and 15–21 µm.

Figure 4. Characterization of porous titanium with biomimetic coating.

(A) SEM image of the precipitate on sample surfaces soaked in SBF for 14 days. (B). TF-XRD pattern of the sample surface after alkali-heat treatment and subsequently immersion in SBF for 14 days. (C) FTIR spectra of chemically pretreated Ti6Al4V samples soaked in SBF for 14 days.

Figure 5. SEM morphologies of cells on porous titanium samples after 14 days of culture.

A great number of osteoblasts attached to the (A–B) pure porous titanium scaffolds and (C–D) porous titanium scaffolds with biomimetic coating, and presented an elongated morphology with cytoplasmic extensions on scaffolds. There were no obvious differences in cell adhesion and morphology between the uncoated and coated samples.

Figure 6. H&E stained sections of sample after 14 days of in vitro culture.

Large amount of extracellular matrix deposited among the cells, and some cells migrated into the inner pore of both (A–B) uncoated and (C–D) coated samples.

Figure 7. Cell viability of the sample over 14 days of in vitro culture.

The cells on the porous titanium exerted a high and sustained proliferation rate within 14 days, and no significant difference was observed between the uncoated and coated samples at each timepoint (P>0.05). TiI, pure porous titanium implant; TiC, porous titanium implant with biomimetic coating.

Figure 8. Fluorochrome labeling of bone regeneration at 12 weeks post-surgery.

The fluorescent labeling indicated that abundant new bone growth into the porous titanium and continuous process of bone remodeling in both (A–B) uncoated and (C–D) coated samples. (E) The rate of bone mineralization apposition was similar in these two kinds of porous titanium at 12 weeks post-surgery (P>0.05). TiI, pure porous titanium implant; TiC, porous titanium implant with biomimetic coating.

Figure 9. Histological staining for osteogenesis within porous titanium after implantation for 4, 8 and 12 weeks.

The images showed that the rapid increase of bone ingrowth into the pores of titanium throughout the experiment and close contact between bone tissue and EBM implants at 12 weeks post-surgery, while there were no obvious differences between the uncoated and coated samples at each timepoint. TiI, pure porous titanium implant; TiC, porous titanium implant with biomimetic coating.

Figure 10. Histomorphometric analysis of new bone formation.

The data showed a high percentage of newly formed bone in the defect region in both coated and uncoated implants during the whole experiment (P>0.05). TiI, pure porous titanium implant; TiC, porous titanium implant with biomimetic coating.