Sintered porous Ti6Al4V scaffolds incorporated with recombinant human bone morphogenetic protein-2 microspheres and thermosensitive hydrogels can enhance bone regeneration

Abstract

A well-controlled powder sintering technique was used to fabricate porous Ti6Al4V scaffold. The thermosensitive chitosan thioglycolic acid (CS-TA) hydrogel was used as a carrier to inject recombinant human bone morphogenetic protein-2 (rhBMP-2) microspheres into pores of the Ti6Al4V scaffold at 37 °C, and then the porous Ti6Al4V/rhBMP-2 loaded hydrogel composite was obtained. The bare Ti6Al4V scaffold was used as the control. The characteristics and mechanical properties of the scaffold, rheological properties of the hydrogels and the rhBMP-2 loaded hydrogel, the release of the rhBMP-2 loaded hydrogel, and the biological properties of the two types of samples were evaluated by in vitro and in vivo tests. Results indicated that the sintered porous Ti6Al4V had high porosity, large pore size with good mechanical properties. The hydrogel and rhBMP-2 loaded hydrogel showed thermosensity. The rhBMP-2 loaded hydrogel showed a stable and extended release profile without too high burst release of rhBMP-2. Both groups showed good biocompatibility and osteogenic ability. However, according to the results of cell tests and implantation, the group with rhBMP-2 loaded hydrogel had significantly higher cell proliferation rate, faster bone growth speed, and more bone ingrowth at every time point. Therefore, the sintered porous Ti6Al4V scaffolds incorporated with rhBMP-2 microspheres and CS-TA hydrogel was effective in enhancing the bone regeneration, and prospects a good candidate for application in orthopedics.

Fig. 1. Schematic illustration of the preparation of the porous Ti6Al4V and rhBMP-2 loaded hydrogel system.

Fig. 2. Characterization of the sintered porous Ti6Al4V scaffold: (a) is cross-section images obtained by micro-CT, the scale bar represents 500 μm; (b) is the SEM image, and scale bar represents 1 mm.

Fig. 3. (a) Temperature dependence of storage modulus G′ and loss modulus G′′ of the CS-TA hydrogel; (b) temperature dependence of storage modulus G′ and loss modulus G′′ of the rhBMP-2 loaded hydrogel; (c) in vitro release of rhBMP-2 microspheres and the rhBMP-2 loaded hydrogel.

Fig. 4. Cell proliferation and live/dead fluorescence microscopy images after of the two groups: (a) bare Ti6Al4V scaffold and (c) Ti6Al4V/rhBMP-2-loaded hydrogel composite; and the SEM images of cell attachment: (b) bare Ti6Al4V scaffold and (d) Ti6Al4V/rhBMP-2-loaded hydrogel composite. Scale bars in a and c represent 50 μm; scale bars in b and d represent 100 μm.

Fig. 5. The CCK-8 results of cell proliferation and the expression of ALP, Runx-2 and Col I in the bare Ti6Al4V group and Ti6Al4V/rhBMP-2-loaded hydrogel composite group.

Fig. 6. Fluorescence images of the two groups: (a) bare Ti6Al4V scaffold and (b) Ti6Al4V/rhBMP-2-loaded hydrogel composite. Bone mineralization apposition rate is a vertical space between two fluorochrome markers interval (space between the white arrows). Scale bars represent 30 μm; histological results after implantation of 4 weeks in the two groups with H&E staining: (c) bare Ti6Al4V scaffold and (d) Ti6Al4V/rhBMP-2-loaded hydrogel composite. Scale bars represent 300 μm.

Fig. 7. The values of (a) MAR and (b) BIC in the two groups at 1, 4 and 8 weeks after implantation. *, P < 0.05. MAR, mineralization apposition rate. BIC, bone-implant contact.