背景

临界尺寸骨缺损因自愈能力有限和修复周期长，常导致再生不完全或功能丧失，传统修复方法如金属植入、自体或异体移植等存在供体损伤、疾病传播及骨整合不良等局限性。人工合成骨移植物虽提供结构支撑，但血管化不足和骨诱导活性受限使其修复效果欠佳。传统骨组织工程采用“自上而下”策略，虽能提供物理支撑，但细胞分布不均、营养扩散不足及微结构仿生性差等问题限制了其应用。近年来，再生医学结合发育生物学提出“发育工程”理念，通过模拟胚胎发育过程构建骨类器官，为骨再生提供了新思路。骨类器官通过细胞自组织形成仿生三维结构，兼具天然骨的生物学特性，显著提升了骨再生的可控性和功能性。然而，干细胞分化控制不足、体外培养周期长及血管化需求仍是骨类器官应用的瓶颈。引入促成骨因子和预血管化策略成为解决这些问题的有效途径，为临界尺寸骨缺损的修复提供了全新解决方案。

研究目的

本课题旨在开发体外构建预血管化骨类器官的有效方法，并结合3D打印技术实现快速原位颅骨缺损修复。具体研究目标包括：

1）优化促成骨微粒的选择与应用，筛选最具成骨潜力的微粒类型并明确其制备工艺与培养条件；

2）构建预血管化骨类器官，结合血管内皮细胞与最优促成骨微粒，开发体外构建方法并评估其成血管与成骨性能；

3）解析促成骨微粒的分子机制，揭示其对成骨分化的调控路径与关键机制；

4）制备复合生物墨水并实现3D生物打印，开发适用于包载预血管化骨类器官的水凝胶材料，构建精准匹配骨缺损结构的组织模型；

5）评估体内修复效果及作用机制，通过体内实验验证预血管化骨类器官在原位颅骨缺损修复中的效果及临床应用潜力。

研究方法

本研究采用以下方法实现预血管化骨类器官的构建及其在颅骨缺损修复中的应用，具体研究方法包括：

1）促成骨微粒筛选与骨类器官构建：以脂肪来源间充质干细胞（MSCs）、人脐静脉内皮细胞（HUVECs）与促成骨微粒（MPs）按1:1:1的比例混合，通过超低附着板结合离心法形成微球体。通过PCR和免疫荧光染色评估成骨基因表达及细胞活性，筛选出最具成骨潜力的微粒;

2）预血管化骨类器官性能评估：通过茜素红（ARS）和碱性磷酸酶（ALP）染色评估成骨性能，利用扫描电镜（SEM）和透射电镜（TEM）观察微观结构；通过体外成血管实验量化血管生成能力，明确HUVECs与促成骨微粒的协同作用；

3）分子机制解析：采用RNA-Seq技术分析差异表达基因，结合Gene Ontology和KEGG数据库富集分析，揭示促成骨微粒调控成骨分化的关键信号通路;

4）3D生物打印与组织体构建：筛选并优化明胶甲基丙烯酰化物（GelMA）水凝胶浓度（5 wt%-10 wt%），加载预血管化骨类器官进行3D生物打印构建可用于骨缺损填充的组织体，观察组织内骨类器官的长期形态变化;

5）体内修复效果验证：通过小鼠颅骨缺损模型，对比预血管化骨类器官组、预血管化聚集体组、干细胞微粒聚集体组及空白对照组的修复效果。利用Micro-CT、组织学染色（H&E、Masson）、免疫荧光染色（OCN、CD31）及MRI扫描，评估矿化组织形成、血管生成及炎症调节效果，验证其临床应用潜力。

研究结果

本研究成功构建了预血管化骨类器官并验证了其在颅骨缺损修复中的应用效果，具体结果如下：

1）促成骨微粒筛选与骨类器官构建：筛选出氧化石墨烯（GO）为最具成骨潜力的微粒，并通过qRT-PCR和免疫荧光染色证实其显著促进成骨基因表达及细胞活性;

2）预血管化骨类器官性能评估：ARS和ALP染色表明加载GO的预血管化骨类器官具有优异的成骨性能；SEM和TEM观察显示GO与细胞紧密结合；体外成血管实验证实HUVECs与GO协同促进血管生成；

3）分子机制解析：RNA-Seq结合Gene Ontology和KEGG分析揭示GO通过PI3K/Akt和Focal Adhesion信号通路调控成骨分化；

4）3D生物打印与组织体构建：筛选7.5 wt% GelMA水凝胶，成功加载预血管化骨类器官并完成3D生物打印，构建骨缺损填充组织体，长期培养显示类器官间相互连接；

5）体内修复效果验证：颅骨缺损模型的Micro-CT分析显示，预血管化骨类器官组在成骨效果上显著优于预血管化聚集体组、干细胞微粒聚集体组及空白对照组；组织学染色、免疫荧光染色及磁共振成像结果进一步证实，预血管化骨类器官在血管生成及炎症调节方面表现出显著的修复效果。

研究结论

本研究提出了一种结合预血管化骨类器官与3D生物打印技术的新策略，用于快速促进原位颅骨再生。基于发育工程原理，成功构建了具有自组织微血管生成和增强成骨分化特性的预血管化骨类器官，作为功能性血管化骨单元。该策略利用3D打印技术克服了传统支架方法的局限性，能够精确复制骨骼复杂结构并确保细胞均匀分布。通过将预血管化骨类器官整合至水凝胶中制备活性生物墨水，实现了高细胞密度、功能性脉管系统和仿生骨组织的构建。实验结果表明，该策略通过模拟干细胞聚集、结合成骨颗粒和促进预血管化，显著加速了原位颅骨缺损的修复。这一创新策略为颅骨缺损治疗提供了高效解决方案，并推动了生物制造与组织工程在再生医学领域的进一步发展。

Background

Critical-sized bone defects, due to their limited self-healing capacity and prolonged repair cycles, often result in incomplete regeneration or functional loss. Traditional repair methods, such as metal implants, autografts, and allografts, face limitations including donor site morbidity, risk of disease transmission, and poor osseointegration. Although synthetic bone grafts provide structural support, insufficient vascularization and limited osteoinductive activity hinder their repair efficacy. Conventional bone tissue engineering employs a "top-down" strategy, offering physical support but suffering from uneven cell distribution, inadequate nutrient diffusion, and poor biomimicry of microstructures, which restricts its application. In recent years, regenerative medicine has integrated developmental biology principles to propose the concept of "developmental engineering," providing new insights into bone regeneration by simulating embryonic development processes to construct bone organoids. Bone organoids, formed through cell self-organization, exhibit biomimetic three-dimensional structures and biological properties akin to natural bone, significantly enhancing the controllability and functionality of bone regeneration. However, challenges such as insufficient control over stem cell differentiation, prolonged in vitro culture periods, and vascularization demands remain bottlenecks for the application of bone organoids. The introduction of osteogenic factors and prevascularization strategies has emerged as an effective approach to address these issues, offering a novel solution for the repair of critical-sized bone defects.

Objective

The objective of this study is to develop an effective method for the in vitro construction of prevascularized bone organoids and to achieve rapid in situ repair of cranial bone defects using 3D printing technology. The specific research goals include: 

1) Optimizing the selection and application of osteogenic microparticles by screening the most osteogenic potential particle types and defining their preparation processes and suitable culture conditions; 

2) Constructing prevascularized bone organoids by combining vascular endothelial cells with optimal osteogenic microparticles, developing in vitro construction methods, and evaluating their vascularization and osteogenic performance; 

3) Elucidating the molecular mechanisms of osteogenic microparticles by revealing their regulatory pathways and key mechanisms in osteogenic differentiation at the molecular level; 

4) Preparing composite bioinks and achieving 3D bioprinting by developing hydrogel materials suitable for encapsulating prevascularized bone organoids and constructing tissue models that precisely match bone defect structures; 

5) Evaluating in vivo repair effects and mechanisms by conducting in vivo implantation experiments to validate the efficacy and clinical potential of prevascularized bone organoids in the repair of in situ cranial bone defects.

Methods

This study employs the following methods to achieve the construction of prevascularized bone organoids and their application in cranial bone defect repair. The specific research methods include: 

1) Screening of Osteogenic Microparticles and Construction of Bone Organoids: Adipose-derived mesenchymal stem cells (MSCs), human umbilical vein endothelial cells (HUVECs), and osteogenic microparticles (MPs) were mixed at a ratio of 1:1:1. Microspheres were formed using ultra-low attachment plates combined with centrifugation, and culture conditions were optimized using a dynamic culture system. The most osteogenic microparticles were screened by evaluating osteogenic gene expression and cell activity through qRT-PCR and immunofluorescence staining.

2) Evaluation of Prevascularized Bone Organoid Performance: Assess osteogenic performance using Alizarin Red S (ARS) and alkaline phosphatase (ALP) staining, and observe microstructures using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Quantify vascularization capability through in vitro angiogenesis experiments to clarify the synergistic effects of HUVECs and osteogenic microparticles. 

3) Elucidation of Molecular Mechanisms: Analyze differentially expressed genes (DEGs) using RNA-Seq technology, and perform enrichment analysis with Gene Ontology and KEGG databases to reveal key signaling pathways regulated by osteogenic microparticles in osteogenic differentiation. 

4) 3D Bioprinting and Tissue Construction: Screen and optimize the concentration of gelatin methacryloyl (GelMA) hydrogel (5 wt%-10 wt%) to load prevascularized bone organoids for 3D bioprinting, constructing tissue constructs for defect filling. Observe long-term morphological changes of the organoids within the constructs. 

5) In Vivo Repair Efficacy Validation: Micro-CT analysis of the cranial bone defect model demonstrated that the prevascularized bone organoid group exhibited significantly superior osteogenic effects compared to the prevascularized aggregate group, stem cell-microparticle aggregate group, and blank control group. Histological staining and immunofluorescence staining further confirmed that the prevascularized bone organoids showed remarkable repair effects in terms of vascularization and inflammation modulation.

Outcomes

This study successfully constructed prevascularized bone organoids and validated their application in cranial bone defect repair. The specific results are as follows: 

1) Screening of Osteogenic Microparticles and Construction of Bone Organoids: Graphene oxide (GO) was identified as the most osteogenic microparticle, and its significant promotion of osteogenic gene expression and cell activity was confirmed through qRT-PCR and immunofluorescence staining. 

2) Evaluation of Prevascularized Bone Organoid Performance: ARS and ALP staining demonstrated that GO-loaded prevascularized bone organoids exhibited excellent osteogenic performance. SEM and TEM observations revealed close integration between GO and cells. In vitro angiogenesis experiments confirmed the synergistic promotion of vascularization by HUVECs and GO.

3) Elucidation of Molecular Mechanisms: RNA-Seq combined with Gene Ontology and KEGG analysis revealed that GO regulates osteogenic differentiation through the PI3K/Akt and Focal Adhesion signaling pathways. 

4) 3D Bioprinting and Tissue Construct Construction: The 7.5 wt% GelMA hydrogel was selected to successfully load prevascularized bone organoids and complete 3D bioprinting, constructing tissue constructs for bone defect filling. The long-term culture showed interconnected organoids within the constructs. 

5) In Vivo Repair Efficacy Validation: Micro-CT analysis of a mouse cranial bone defect model demonstrated that the prevascularized bone organoid group exhibited significantly superior osteogenic effects compared to the pre-vascularized aggregate group, stem cell microparticle aggregate group, and blank control group. Histological staining,  immunofluorescence staining, and magnetic resonance imaging results further confirmed the significant repair effects of prevascularized bone organoids in vascularization and inflammation modulation.

Conclusion

This study proposes a novel strategy combining prevascularized bone organoids with 3D bioprinting technology to rapidly promote in situ cranial bone regeneration. Based on developmental engineering principles, prevascularized bone organoids with self-organized microvascular generation and enhanced osteogenic differentiation capabilities were successfully constructed as functional vascularized bone units. This strategy overcomes the limitations of traditional scaffold-based methods by utilizing 3D printing technology to precisely replicate complex bone structures and ensure uniform cell distribution. By integrating prevascularized bone organoids into hydrogels to prepare bioactive bioinks, the construction of high cell density, functional vascular systems, and biomimetic bone tissues was achieved. Experimental results demonstrate that this strategy significantly accelerates the repair of in situ cranial bone defects by simulating stem cell aggregation, incorporating osteogenic particles, and promoting prevascularization. This innovative approach provides an efficient solution for cranial bone defect treatment and advances the development of biomanufacturing and tissue engineering in the field of regenerative medicine.