Tricarboxylic Acid Cycle Metabolite-Coordinated Biohydrogels Augment Cranial Bone Regeneration Through Neutrophil-Stimulated MSCs Recruitment and Histone Acetylation-Mediated Osteogenesis [RNA-Seq]

Cranial bone defect is a major clinical challenge that increases the life burden of patients. Tricarboxylic acid (TCA) cycle metabolites have recently drawn considerable attention in the field of bone tissue regeneration due to their bio-safety, low cost, structural stability, and effectiveness. However, the development of TCA cycle metabolite-modified biomimetic grafts for skull bone regeneration via a facile and general approach remains lacking. Moreover, the mechanism underlying the release of TCA cycle metabolites from biomaterials in immune responses and mesenchymal stem cells (MSCs) fate (migration and differentiation) remain unknown. Herein, inspired by the Hofmeister effects, we developed a series of TCA metabolite (in the form of sodium salt)-coordinated biomimetic hydrogels (CGG) with strong mechanical and anti-swelling performances. Importantly, the sodium citrate (Na3Cit)-treated CGG hydrogels (CGG-Cit) with the highest mechanical modulus and strength significantly promoted skull bone regeneration in rats and mice. Mechanistically, using a transgenic mouse model, bulk RNA sequencing, and single-cell RNA sequencing, we demonstrated that CGG-Cit promotes Gli1+ MSCs migration via neutrophil-secreted OSM. Our results also indicate that CGG-Cit improved osteogenesis via enhanced H3K9ac modification on osteogenic master genes. Taken together, the immune microenvironments and MSCs fate-regulated biomimetic hydrogels developed herein represent a highly efficient and facile approach toward skull bone tissue regeneration with great potential for bench-to-bedside translation. To assess the molecular responses to CGG-Cit hydrogels and CGG hydrogels in calvarial bone defects, rats were sacrificed at week 4 post-surgery. The bone defect areas were carefully trimmed and minced to pieces, and total RNA was extracted using Trizol Reagent (Invitrogen, Thermo Fisher Scientific, USA) and subjected to RNA sequencing.

颅骨缺损（Cranial bone defect）是一项重大临床挑战，会加重患者的生活负担。近年来，三羧酸循环（Tricarboxylic acid, TCA）代谢物因其生物安全性、低成本、结构稳定性与有效性，在骨组织再生领域受到广泛关注。然而，采用简便通用的方法开发用于颅骨骨再生的TCA循环代谢物修饰仿生移植物的研究仍属空白。此外，TCA循环代谢物从生物材料中释放，进而调控免疫应答与间充质干细胞（mesenchymal stem cells, MSCs）命运（迁移与分化）的潜在机制仍未阐明。
有鉴于此，受霍夫麦斯特效应（Hofmeister effects）启发，本研究开发了一系列以钠盐形式存在的TCA代谢物配位仿生水凝胶（CGG），该材料具备优异的力学性能与抗溶胀特性。尤为关键的是，经柠檬酸钠（Na₃Cit）修饰的CGG水凝胶（CGG-Cit）拥有最高的力学模量与强度，可显著促进大、小鼠的颅骨骨再生。
从机制层面分析，本研究借助转基因小鼠模型、整体转录组测序与单细胞转录组测序，证实CGG-Cit可通过中性粒细胞分泌的抑瘤素M（OSM）促进Gli1+间充质干细胞的迁移。研究结果同时表明，CGG-Cit可通过增强成骨关键基因的H3K9ac组蛋白修饰，促进成骨分化。
综上，本研究开发的可调控免疫微环境与间充质干细胞命运的仿生水凝胶，为颅骨骨组织再生提供了一种高效简便的策略，具备良好的实验室到临床转化潜力。
为评估CGG-Cit水凝胶与纯CGG水凝胶对颅骨缺损的分子调控效应，研究团队于术后第4周处死大鼠。仔细修剪并剪碎骨缺损区域的组织，采用Trizol试剂（Invitrogen，赛默飞世尔科技，美国）提取总RNA并进行转录组测序。