The paradoxical effect of cobalt on biological processes has aroused controversy regarding the application of cobalt-based biomaterials in bone regeneration. Tuning the dose range of cobalt ions may be a valid strategy to resolve the controversies about cobalt use for orthopedic applications. Recent progress in bone biology has highlighted the effects of multisystem cooperation (especially of osteoimmune, skeletal, and vascular systems) on bone dynamics. Before the application of this dose-tuning strategy, a deeper understanding of its dose-dependent effect on the cooperation of osteoimmune, skeletal, and vascular systems is needed. However, due to the difficulties with investigating the interaction of multiple systems in vitro, the multimodal effects of cobalt on bone homeostasis were investigated here, in an in vivo scenario.

Methods: In vitro CCK8 assay and cytoskeletal staining were preformed to detecte the cell cytotoxic reaction in response to 0.1-100 ppm cobalt stimulation. Blood clot containing 0.1 to 5 ppm of cobalt were implanted in the rat calvarium defect. The gene profile of osteoimmune, skeletal, and vascular system as well as the systemic toxicity were evaluated via RT-qPCR, histological analysis and inductively coupled plasma mass spectrometry. The bone regeneration, osteoclastogenesis and vascularization were assessed by micro-ct and histological analysis.

Results: Cobalt concentration below 5 ppm did not cause cell toxicity in vitro. No systemic toxicity was observed in vivo at 0.1-5 ppm cobalt concentration. It was found that the early cytokine profiles of the multiple interacting systems were different in response to different cobalt doses. Most of the anti-inflammatory, osteogenic, and proangiogenic factors were upregulated in the 1 ppm cobalt group at the early stage. In the late stage, the 1ppm group was most superior in bone regenerative effect while the 5 ppm group displayed the strongest osteoclastogenesis activity.

Conclusions: The 1 ppm concentration of cobalt yielded the most favorable cooperation of the osteoimmune, skeletal, and vascular systems and subsequently optimal bone regeneration outcomes. Tuning the cobalt dose range to manipulate the cooperation of osteoimmune, skeletal, and vascular systems could be a promising and valuable strategy to prevent paradoxical effects of cobalt while preserving its beneficial effects.

钴对生物过程的反常效应引起了关于钴基生物材料在骨再生中的应用的争议。调整钴离子的剂量范围可能是解决有关骨科应用中使用钴的争议的有效策略。骨骼生物学的最新进展突显了多系统合作（尤其是骨免疫，骨骼和血管系统）对骨骼动力学的影响。在应用这种剂量调整策略之前，需要更深入地了解其对骨免疫，骨骼和血管系统协同作用的剂量依赖性作用。但是，由于在体外研究多个系统之间的相互作用存在困难，在骨稳态钴多影响在这里考察，在体内的情况。

方法：进行体外CCK8检测和细胞骨架染色以检测0.1-100 ppm钴刺激下的细胞毒性反应。将含有0.1至5 ppm钴的血凝块植入大鼠颅骨缺损中。通过RT-qPCR，组织学分析和电感耦合等离子体质谱法评估骨免疫，骨骼和血管系统的基因谱以及全身毒性。通过micro-ct和组织学分析评估骨再生，破骨细胞生成和血管形成。

结果：钴浓度低于5 ppm不会在体外引起细胞毒性。钴浓度为0.1-5 ppm时，在体内未观察到全身毒性。已经发现，多种相互作用系统的早期细胞因子谱响应于不同的钴剂量而不同。在早期阶段，在1 ppm钴组中，大多数抗炎，成骨和促血管生成因子均被上调。在晚期阶段，1ppm组的骨再生效果最佳，而5ppm组则表现出最强的破骨细胞生成活性。

结论：1 ppm的钴浓度产生了骨免疫，骨骼和血管系统的最有利的配合，并随后获得了最佳的骨再生结果。调节钴的剂量范围以控制骨免疫，骨骼和血管系统的合作可能是一种有希望且有价值的策略，可以在保持钴的有益效果的同时防止钴的矛盾效应。