创伤、感染和肿瘤切除引起的临界大小颅骨缺损对高效骨替代物提出了巨大需求。在此，我们成功通过多步骤程序组装了一种混杂交联分级微孔水凝胶支架（PHCLS），该程序包括（i）聚乳酸-共-聚乙醇酸/纳米羟基磷灰石（PLGA-HAP）多孔微球的制备，（ii）将微球嵌入多巴胺修饰的透明质酸和胶原I（Col I）溶液中，并通过多巴胺多酚与（i）Col I氨基（通过迈克尔加成）和（ii）PLGA-HAP（通过钙离子螯合）结合进行交联。引入PLGA-HAP不仅提高了矩阵内孔隙尺寸和孔隙通讯的多样性，还显著增强了压缩强度（5.24倍，77.5 kPa）和降解性能，以构建更稳定的机械结构。特别是，PHCLS（200 mg，nHAP）在体外促进了骨髓间充质干细胞的增殖、渗透和血管分化，以及显著的异位血管生成和矿化，在30天后存储模量增强了2.5倍。同时，适当的基质微环境通过加速内源干细胞在体内的招募启动了血管生成和早期成骨作用。总体而言，PHCLS在兔颅骨缺损模型中实现了大幅颅骨重建，在植入后12周与天然颅骨相比，达到了85.2%的破裂负荷强度和84.5%的骨体积分数。总之，该研究揭示了分级微孔水凝胶支架为颅骨缺损治疗提供了有希望的策略。

Critical-size skull defects caused by trauma, infection, and tumor resection raise great demands for efficient bone substitutes. Herein, a hybrid cross-linked hierarchical microporous hydrogel scaffold (PHCLS) was successfully assembled by a multistep procedure, which involved (i) the preparation of poly(lactic-co-glycolic)/nanohydroxyapatite (PLGA-HAP) porous microspheres, (ii) embedding the spheres in a solution of dopamine-modified hyaluronic acid and collagen I (Col I) and cross-linking via dopamine polyphenols binding to (i) Col I amino groups (via Michael addition) and (ii) PLGA-HAP (via calcium ion chelation). The introduction of PLGA-HAP not only improved the diversity of pore size and pore communication inside the matrix but also greatly enhanced the compressive strength (5.24-fold, 77.5 kPa) and degradation properties to construct a more stable mechanical structure. In particular, the PHCLS (200 mg, nHAP) promoted the proliferation, infiltration, and angiogenic differentiation of bone marrow mesenchymal stem cells in vitro, as well as significant ectopic angiogenesis and mineralization with a storage modulus enhancement of 2.5-fold after 30 days. Meanwhile, the appropriate matrix microenvironment initiated angiogenesis and early osteogenesis by accelerating endogenous stem cell recruitment in situ. Together, the PHCLS allowed substantial skull reconstruction in the rabbit cranial defect model, achieving 85.2% breaking load strength and 84.5% bone volume fractions in comparison to the natural cranium, 12 weeks after implantation. Overall, this study reveals that the hierarchical microporous hydrogel scaffold provides a promising strategy for skull defect treatment.