Abstract
2.1.1 Preparation of drug-laden Gel/HA composite
Alendronate (ALN), is a commonly used bisphosphonate drug for osteoporosis management and bone regeneration. [33] However, direct loading of ALN onto Gel often results in burst drug release due to the fast Gel degradation in vivo. In contrast, degradable HA microsphere is an excellent drug delivery vehicle, which can provide sustained/controlled drug release as well as promoting osteoblasts mineralization/maturation (due to slow release of Ca2+ and PO43-). [34] To prepare the ALN-laden HA microspheres/Gel composite (ALN-HA/Gel), HA microspheres were first synthesized following established procedure. [35] 0.5 g ALN was then added into 50 mL aqueous dispersion containing 2.0 g HA microspheres. After mixing using shaking incubator under 37 ℃ for 48 h, ALN-HA were collected by centrifugation and vacuum dried at 40 ℃ for 24 h. ALN concentration in the supernatant solution was evaluated by UV-vis spectroscopy. The drug-laden Gel/ALN-HA can be obtained by dispersing 3.0g ALN-HA microspheres in 50 mL deionized water, followed by addition of 8.0 g Gel, and stirred magnetically at 1000 rpm under 50 ℃ for 1 h.
2.1.2 Preparation of 3D printed Ti6Al4V scaffolds
3D printed Ti6Al4V scaffolds (named as Ti) were manufactured by electron beam melting (EBM) facility in-house. The lattice structure was designed by computer-assisted design (CAD) software (Magics, Materialise, Belgium) based on a dodecahedron unit cell with strut diameter of 300 μm and porosity of 80%. Cubic scaffolds (5mm × 5mm × 5mm) were used for in vitro testing and cylindrical scaffolds (Φ = 6mm, height = 6mm) were used for in vivo experiments. All samples were thoroughly cleaned by sonication in acetone, alcohol and deionized water for 30 min, respectively. The Ti scaffolds were subsequently subjected to hydrothermal treatment. [36] Specifically, the scaffolds were immersed in an aqueous solution (m(H2O2):m(H3PO4) 9:1) and placed in a Teflon-lined autoclave under 220 ℃ for 24 h. After the treatment, scaffolds were washed with distilled water and dried in air at room temperature for 24 h. The hydrothermally treated samples were named as HR-Ti.
2.1.3 Preparation of Gel/ALN-HA infused HR-Ti scaffold
The Gel/ALN-HA mixture was infused into the HR-Ti, then the infused scaffolds were held at -80 ℃ overnight followed by freeze-drying at -40 ℃ for 48 h. After that, the infused scaffolds were immersed in 100 mL ethanol containing 50mmol/L 1-ethyl-3-(3- (dimethylamino) propyl) carbodiimide hydrochlorid (EDC∙HCl) and N-hydroxysuccinimide (NHS) at 4 ℃ for 10 h to crosslink the Gel content. Afterwards, the scaffolds were washed three times by ethanol and freeze-dried again. The final scaffolds were named as Gel/ALN-HA/HR-Ti. For comparison, ALN/HR-Ti (without Gel and HA), Gel/ALN/HR-Ti (without HA) and Gel/HA/HR-Ti (without ALN) were also prepared.
