研究背景：骨缺损是临床治疗中的一大挑战，当骨缺损体积超越了机体自身的再生能力时，需依赖额外的干预措施。当前，骨缺损主流的治疗策略包括自体/异体骨移植，但由于骨移植存在来源有限、供区并发症和排异反应等问题，人工合成生物填充材料的应用逐渐引起关注。电刺激材料是一种新兴的骨填充生物材料，其可通过产生局部电刺激促进骨缺损修复。然而，现有的用于骨缺损修复的电刺激生物材料仍存在生物降解和生物效能等方面的诸多不足，具有极大的改进空间。压电材料是电刺激材料的一种，具有诱发电刺激方式便捷的优势。甘氨酸晶体作为压电材料的一种，具备完全生物可降解的能力。导电水凝胶材料可用作电刺激材料中的另一组分，在促进电刺激信号传导的同时可增加电刺激与细胞的接触，同时适合硬组织缺损的填充，依据其导电成分的不同可分为基于离子或基于电子的导电水凝胶。相较于传统的基于导电聚合物的电子导电水凝胶，以锂离子作为主要导电成分的离子导电水凝胶理论上可在增加水凝胶导电性的同时改善材料对细胞促成骨和促成血管的能力。因此，通过开发压电-Li⁺离子导电水凝胶复合体，有望获得一种更理想的新型骨缺损修复材料。

研究目的：本研究拟通过静电纺丝技术制备甘氨酸晶体-聚己内酯压电层，并与含锂离子的可注射导电水凝胶层粘合，构建一种完全可生物降解的、可由超声激发产生交流电刺激的压电-Li⁺离子导电水凝胶复合体。研究拟通过体外和体内实验验证其促成骨和促成血管性能以及对骨缺损的修复能力，并探索其成骨相关机制。

研究方法：利用静电纺丝技术制备甘氨酸晶体-聚己内酯压电膜，与Li⁺离子导电水凝胶粘合制备新型压电-Li⁺离子导电水凝胶复合体（Piezo-ionic hydrogel assembly，PIHA），与基于电子的导电水凝胶粘合获得压电-电子导电水凝胶复合体（Piezo-electronic hydrogel assembly，PEHA），对材料的表面形貌、物化性能进行表征。通过体外实验观察材料的细胞、血液相容性和促骨髓间充质干细胞增殖、迁移、成骨分化性能，以及促骨微血管内皮细胞成管性能，并利用转录组学测序技术、实时荧光定量聚合酶链式反应、蛋白质免疫印迹技术探索材料促骨髓间充质干细胞成骨能力的相关分子机制。通过Micro-CT、H&E染色、Masson染色、免疫组化染色评估材料对大鼠颅骨临界骨缺损的修复能力以及组织相容性。

研究结果：在超声刺激下，本研究制备的甘氨酸晶体-聚己内酯压电膜可产生良好的交流电刺激，经导电水凝胶传导至细胞发挥作用。经测试，研究制备的压电层的压电系数d₃₃达到约17.1 pC/N，Li⁺离子导电水凝胶的电导率达到约3.1 S/m，高于基于电子的导电水凝胶，PIHA的电压输出大小可达到约260mV。经体外实验测试，PIHA和PEHA材料均具有良好的细胞相容性和血液相容性，BMSCs细胞与材料共培养时铺展良好，材料溶血率均低于5%。在超声激发下，PIHA材料较PEHA材料表现出更强的体外促骨髓间充质干细胞增殖、成骨分化和迁移能力，以及促骨微血管内皮细胞成管能力。转录组学、实时荧光定量聚合酶链式反应、蛋白质免疫印迹技术实验结果初步表明，超声激发PIHA产生电刺激后，激活了骨髓间充质干细胞内钙离子相关信号通路、PI3K-AKT信号通路以及Wnt/β-catenin信号通路，促进了Runx2、OCN、OPN这些成骨相关基因和蛋白的表达。在体内实验中，Micro-CT证实了将PIHA材料植入并进行超声刺激8周后，大鼠颅骨临界骨缺损新生骨骨密度、骨体积及骨体积分数较对照组显著增加，H&E染色、Masson染色证实了新生骨的成熟度较对照组高，免疫组化染色证实了其成骨组织内相关蛋白OCN、OPN的表达量较对照组高，此外，对大鼠的心肝脾肺肾组织切片染色结果证明PIHA材料具有良好的组织相容性，无系统性毒性。

研究结论：本研究开发了一种新型的压电-Li⁺离子导电水凝胶复合体，其压电层可在超声激发下产生交流电刺激，其导电水凝胶具备良好的导电性能，且内含的锂离子可以与电信号共同对细胞产生刺激，同时可注射性导电水凝胶有利于对骨缺损部位填充，简化干预流程。该材料细胞、组织相容性良好，可促进细胞增殖、迁移、成骨和成血管能力，用于骨缺损修复效果显著。通过对促成骨机制的初步探索证实了压电-Li⁺离子导电水凝胶复合体可激活骨髓间充质干细胞内钙离子相关信号通路、PI3K-AKT信号通路以及Wnt/β-catenin信号通路，促进成骨相关蛋白的表达，为电刺激材料在骨缺损修复中的应用提供了充分的理论依据。

Background: Bone defects pose a significant clinical challenge, especially when the size of the defect exceeds the body’s intrinsic regenerative capacity, which needs additional therapeutic intervention. Currently, the main clinical interventions for bone defect repair include bone autografts and allografts. However, these interventions are limited by issues such as the lack of donor, complications at the harvest site, and immune rejection. As a result, increasing attention has been directed toward using synthetic biomaterials as bone substitutes. Electrical stimulation materials represent a new type of bone repair biomaterials, they are capable of generating localized electrical stimuli to enhance bone regeneration. However, existing electrical stimulation biomaterials still face limitations in terms of biodegradability and biological efficacy, indicating considerable room for improvement. Piezoelectric material is a subclass of electrical stimulation biomaterials, it has the advantage of producing electrical stimuli which can be induced through simple methods. Glycine (Gly) crystals, as a type of piezoelectric material, are fully biodegradable. Conductive hydrogels can serve as another component of electrical stimulation systems. They not only facilitate electrical signal transmission but also enhance the interface between electrical stimuli and cells, while can be filled into irregular bone defects. Depending on their conductive mechanism, conductive hydrogels can be categorized into ion-based and electron-based systems. Compared to traditional electron-conductive hydrogels based on conductive polymers, ion-conductive hydrogels containing lithium ions as the main conductive medium may simultaneously enhance the electrical conductivity and improve the material's pro-osteogenic and pro-angiogenic potential. Therefore, the development of a piezoelectric-Li⁺ ion-conductive hydrogel composite is expected to yield a more ideal and effective material for bone defect repair.

Objective: This study aims to fabricate a fully biodegradable piezoelectric-Li⁺ ion- conductive hydrogel composite capable of generating alternating current (AC) electrical stimulation under ultrasonic activation. The composite is constructed by electrospinning a glycine crystal-polycaprolactone (PCL) piezoelectric layer and integrating it with an injectable lithium ion-conductive hydrogel. The biological effects of the material, including its pro-osteogenic and pro-angiogenic potential and its ability to repair bone defects, will be evaluated through in vitro and in vivo experiments, with further exploration of its osteogenic mechanisms.

Methods: The Gly-PCL piezoelectric nanofiber mat was fabricated using electrospinning. This nanofiber mat was then integrated with a lithium ion-conductive hydrogel to form the novel Piezo-ionic hydrogel assembly (PIHA), and with a PEDOT-based electronic conductive hydrogel to form the Piezo-electronic hydrogel assembly (PEHA). The physical morphology and physicochemical properties of the materials were characterized. In vitro experiments assessed cytocompatibility and hemocompatibility, as well as the materials' ability to promote the proliferation, migration, and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), and the tube formation capacity of bone microvascular endothelial cells (BMECs). Transcriptomic sequencing, RT-qPCR, and Western blot were used to investigate molecular mechanisms of how the materials enhanced BMSCs osteogenic differentiation. The in vivo performance of the materials was evaluated in a rat critical-sized calvarial defect model, micro-CT, H&E staining, Masson staining, and immunohistochemical staining were used to assess bone regeneration and tissue compatibility.

Results: Under ultrasonic stimulation, the Gly-PCL piezoelectric nanofiber mat generated stable alternating current, which was effectively transmitted through the conductive hydrogel to the cells. The d₃₃ of the Gly-PCL piezoelectric nanofiber mat was approximately 17.1 pC/N. The electrical conductivity of the Li⁺ ion-conductive hydrogel reached approximately 3.1 S/m, higher than that of the electronic conductive hydrogel. The voltage output of the PIHA composite reached up to approximately 260 mV. In vitro results demonstrated that both PIHA and PEHA exhibited excellent cytocompatibility and hemocompatibility. BMSCs adhered and spread well when co-cultured with PIHA and PEHA, and the hemolysis rate remained below 5%. Under ultrasound stimulation induced electrical stimuli, PIHA showed better ability in promoting BMSCs proliferation, osteogenic differentiation, and cell migration, as well as enhancing BMECs tube formation. Transcriptomic analysis, RT-qPCR, and Western blot indicated that ultrasound induced electrical stimulation of PIHA led to the upregulation of calcium signaling, PI3K-AKT, and Wnt/β-catenin pathways in BMSCs, promoting the expression of osteogenic markers such as Runx2, OCN, and OPN. In vivo, micro-CT analysis confirmed that PIHA combined with ultrasound stimulation significantly enhanced new bone density, bone volume, and bone volume fraction in the calvarial defect area after 8 weeks. Histological analysis by H&E and Masson staining revealed more mature new bone formation in the PIHA group compared to the control group. Immunohistochemical staining also confirmed higher expression levels of osteogenic proteins OCN and OPN in newly regenerated bone. Additionally, histological examination of major organs (heart, liver, spleen, lung, kidney) revealed no signs of systemic toxicity, demonstrating good biocompatibility of PIHA.

Conclusion: This study developed a novel piezoelectric-Li⁺ ion-conductive hydrogel composite, in which the piezoelectric layer generates AC stimulation under ultrasound, and the conductive hydrogel offers excellent conductivity and biocompatibility. The inclusion of lithium ions provides additional bioactivity, while the injectability and in situ gelation of the hydrogel facilitate defect filling and simplify the surgical procedure. The composite demonstrated excellent compatibility with cells and tissues, significantly promoted cell proliferation, migration, osteogenesis, and angiogenesis, and exhibited effective bone defect repair both in vitro and in vivo. In terms of mechanism, the composite was shown to activate calcium-related, PI3K-AKT, and Wnt/β-catenin signaling pathways in BMSCs, thereby enhancing osteogenic gene and protein expression. These findings provide strong theoretical support for the application of electrically active biomaterials in bone defect repair.