Abstract: Objective To explore the construction of a time-sequential coating system on the surface of 3D-printed titanium alloy implants, which can sequentially deliver dual functions of anti-tumor and osteogenic promotion for the treatment and repair of bone defects following osteosarcoma surgery. Methods Porous Ti-6Al-4V samples were fabricated using selective laser melting(SLM), and the samples were first subjected to flow acid etching treatment(AE group). Subsequently, nano-hydroxyapatite(nHA)was mixed with agarose solution to form an internal hydrogel, which was then infiltrated into the porous structure through mechanical interlocking mechanisms(AN group). Subsequently, the AN group samples were then immersed in a calcium chloride solution to facilitate calcium ion adsorption through hydrogel swelling. Finally, a composite solution of sodium alginate, gelatin, and doxorubicin(DOX)(outer hydrogel)was applied to the surface of the AN group samples via a layer-by-layer crosslinking method(AG group). The surface morphology, roughness, wettability, corrosion resistance and mechanical properties of each group were characterized using scanning electron microscopy(SEM), three-dimensional confocal laser microscopy, contact angle measurement, electrochemical workstation and ME50 electronic universal testing machine. The swelling ratio and degradation rate of the inner and outer hydrogels were determined by the immersion method, and the in vitro release kinetics of DOX were evaluated. Rat bone marrow mesenchymal stem cells(BMSCs)were cultured on the surfaces of the AE and AN groups to evaluate their adhesion, proliferation, and differentiation capabilities. In order to evaluate the antitumor efficacy and toxicity of the samples, human osteosarcoma cells(HOS)and rat bone marrow mesenchymal stem cells were cultured on the surfaces of the AE and AG groups. Tumor-bearing models were established in the subcutaneous tissue of BALB/c mice. In these models, AE and AG implants were surgically positioned at the tumor tissue base. This approach was used to systematically evaluate the in vivo antitumor efficacy and biosafety profiles of the implants. Results SEM analysis revealed the presence of micron-scale pit structures on the surface of the AE group samples, exhibiting uniform distribution. In the AN group samples, agarose and nanohydroxyapatite were clearly observed to be present within the porous structure, forming a large-pore network on the scale of hundreds of micrometers. The AG group samples exhibited a surface with larger micropores, showing wrinkle-like features caused by the loading of DOX, while the interior exhibited a significant number of millimeter-scale porous structures. Surface roughness analysis revealed that the roughness of the AE and AN groups remained within the range of 4-5 μm, while the roughness of the AG group decreased to 2.180 μm. Contact angle measurements demonstrated that the loading of the inner hydrogel significantly enhanced the hydrophilicity of the AE group samples, whereas the loading of the outer hydrogel slightly reduced their hydrophilicity. Electrochemical tests demonstrated that the sequential loading of the double-layer hydrogel led to a substantial enhancement in the corrosion resistance of the samples. Swelling and degradation experiments revealed that the inner hydrogel exhibited a lower swelling ratio and slower degradation rate compared to the outer hydrogel. In vitro drug release kinetics studies indicated that DOX release exhibited an initial burst release profile, with sustained release lasting over 10 days. Cellular experiments demonstrated that, compared to the AE group, the AN group significantly promoted the adhesion, proliferation(P<0.001), and differentiation(alkaline phosphatase, bone morphogenetic protein 2 and osteopontin: P<0.01; osteocalcin: P<0.05)capabilities of rat bone marrow mesenchymal stem cells. In contrast, the AG group exhibited significant killing effects on human osteosarcoma cells(HOS)compared to the AE group(P<0.000 1), while showing only mild toxicity toward BMSCs. Tumor-bearing animal experiments confirmed that the AG group achieved effective antitumor efficacy while maintaining biosafety. Conclusion The double-layer hydrogel is methodically loaded into the 3D-printed porous Ti-6Al-4V implant, and it synergistically delivers dual functions of anti-tumor and osteogenic promotion based on a temporal sequence.

Key words: 3D-printed porous Ti-6Al-4V implant, Double layer hydrogel, Time-sequential, Contributory bone function, Antitumor function