Scaffolds functionalized with bone morphogenetic protein-2 (BMP-2) have shown great potential for bone regeneration. However, structural instability and the necessity for supra-physiological dose have thus far limited practical applications for BMP-2. Protein modification and site-specific covalent immobilization of BMP-2 to carrier materials might be optimal strategies to overcome these problems. Here, we report a broadly applicable strategy where the polyhistidine tag-T4 Lysozyme (His6-T4L) was genetically fused at the N-terminus of BMP-2 and used as a protein spacer, which on one hand enhanced protein solubility and stability, and on the other hand mediated site-specific covalent anchoring of BMP-2 upon binding to nickel-chelated nitrilotriacetic acid (Ni-NTA) microparticles (denoted as MPs-His6-T4L-BMP2) to further maximize its rescued activity. We also constructed a novel gelatin-based hydrogel that was crosslinked by transglutaminase (TG) and tannic acid (TA). This hydrogel, when incorporated with MPs-His6-T4L-BMP2, displayed excellent in-situ injectability, thermosensitivity, adhesiveness and improved mechanical properties. The effective loading mode led to a controlled and long-term sustained release of His6-T4L-BMP2, thereby resulting in enhancement of bone regeneration in a critical-sized bone defect. We believe that the protein modification strategy proposed here opens up new route not only for BMP-2 applications, but can be used to inform novel uses for other macromolecules.

用骨形态发生蛋白-2 (BMP-2) 功能化的支架已显示出巨大的骨再生潜力。然而，结构不稳定性和超生理剂量的必要性迄今为止限制了 BMP-2 的实际应用。BMP-2 的蛋白质修饰和位点特异性共价固定到载体材料上可能是克服这些问题的最佳策略。在这里，我们报告了一种广泛适用的策略，其中多组氨酸标签-T4 溶菌酶（His6-T4L）在 BMP-2 的 N 末端进行基因融合并用作蛋白质间隔物，一方面提高了蛋白质的溶解性和稳定性，另一方面，在与镍螯合次氮基三乙酸 (Ni-NTA) 微粒（表示为 MPs-His6-T4L-BMP2）结合后，介导 BMP-2 的位点特异性共价锚定，以进一步最大化其获救活性。我们还构建了一种新型明胶基水凝胶，该凝胶由转谷氨酰胺酶 (TG) 和单宁酸 (TA) 交联。这种水凝胶，当与 MPs-His6-T4L-BMP2 结合时，表现出优异的原位注射性、热敏性、粘附性和改进的机械性能。有效的加载模式导致 His6-T4L-BMP2 的受控和长期持续释放，从而增强临界尺寸骨缺损的骨再生。我们相信，这里提出的蛋白质修饰策略不仅为 BMP-2 应用开辟了新途径，而且可用于为其他大分子提供新用途。