Recent thrust in the biomaterials research has shifted towards developing bioactive and resorbable scaffolds with nanotechnological interventions. Next-generation bone scaffolds need to be multifunctional with desired osteo-integration ability, conformed resorption rate and in vivo imaging potential. In this study, we synthesized biogenic multifunctional gold nanoparticles (AuNPs) using immobilized crude enzyme (ImCE) of Bacillus licheniformis. The multifunctional characteristics of AuNPs and reusability of ImCE for AuNP synthesis can make the process and application cost-effective. ImCE catalyzed the bioreduction of gold ions to AuNPs of 18.4 ± 3.3 nm. AuNPs showed good hemocompatibility and antioxidant property without any toxic effects. Further, AuNPs demonstrated the increase in alkaline phosphatase activity and calcium deposition through MC3T3-E1 murine preosteoblast cell line and rat bone marrow stromal cells (BMSCs). The osteoinductive effect of AuNPs was also investigated in vivo wherein the composite cryogel scaffold functionalized with bone morphogenic protein-2 (rhBMP-2), zoledronic acid (ZA), and AuNPs was implanted in a muscle pouch of Wistar rats. AuNPs had synergistic effect on bone formation along with rhBMP-2 and ZA as seen from micro-computed tomography and histological analysis. The AuNPs were found to exhibit autofluorescence property and enhanced the degradation of scaffold at the implanted site in vivo. The developed process of using ImCE presents a novel approach for biogenic fabrication of AuNPs with the potential orthopedic application.

Graphical abstract

生物材料研究的最新重点已转向通过纳米技术干预来开发具有生物活性和可吸收性的支架。下一代骨支架需要具有所需的骨整合能力，一致的吸收率和体内成像潜力的多功能。在这项研究中，我们使用地衣芽孢杆菌的固定化粗酶（ImCE）合成了生物多功能金纳米颗粒（AuNPs）。AuNPs的多功能特性和ImCE在AuNP合成中的可重复使用性可以使该方法和应用程序具有成本效益。ImCE催化金离子生物还原为18.4±3.3 nm的AuNP。AuNPs显示出良好的血液相容性和抗氧化性能，没有任何毒性作用。此外，AuNPs通过MC3T3-E1小鼠前成骨细胞细胞系和大鼠骨髓基质细胞（BMSC）证明了碱性磷酸酶活性和钙沉积的增加。还对体内的AuNPs的骨诱导作用进行了研究，其中将用骨形态发生蛋白2（rhBMP-2），唑来膦酸（ZA）和AuNPs功能化的复合冷冻凝胶支架植入Wistar大鼠的肌肉袋中。从微型计算机断层扫描和组织学分析可以看出，AuNP与rhBMP-2和ZA一起对骨形成具有协同作用。发现AuNP在体内植入部位表现出自发荧光性质并增强了支架的降解。使用ImCE的已开发过程为潜在的骨科应用提供了一种生物制备AuNP的新方法。

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