研究背景及目的

股骨头坏死（ONFH）是一种因股骨头血供受损或中断引起的骨坏死性疾病，其发病率、致残率高，严重影响患者生活质量。现有保髋治疗技术在疾病塌陷前期的修复效果有限，尤其对于年轻患者和早期患者，缺乏有效延缓疾病进展甚至避免髋关节置换的策略。因此，本研究创新设计了一种多孔仿生支架，通过物理（Ti）、化学（nHA/col）和生物（BMSCs-exos）的多维模拟，整合力学支撑、成骨诱导和血管再生的功能。通过髓芯减压通道结合GelMa水凝胶将外泌体精准递送至股骨头坏死区域和软骨下骨塌陷部位，实现外泌体长效缓释和骨-血管协同修复，克服传统填充材料强度不足、成骨及成血管能力缺乏的局限，进一步推动外泌体治疗从实验室研究向临床转化，为股骨头坏死早中期保髋治疗提供一定的理论依据与技术支持。

研究方法

本研究通过整合 BMSCs外泌体的提取方法，采用冷冻超速离心法与化学沉淀法双法连提，成功获得高纯度、高产量的外泌体，并通过透射电镜（TEM）、粒径分析（NTA）及标志物（CD9、CD63、CD81）检测对其进行全面表征。在此基础上，利用3D打印激光熔融技术、冷冻干燥法及GelMa水凝胶光固化技术，成功制备负载外泌体的BMSCs-exos@nHA/Col-Ti三维仿生支架，并对其物理、化学及生物性能进行系统表征，包括孔径、孔隙率、降解性能及力学性能等。通过体外实验评估支架对BMSCs增殖、迁移、成骨、成血管能力的促进作用，并利用qPCR检测成骨及成血管相关基因表达。最后，构建兔激素-内毒素诱导的股骨头坏死模型，结合髓芯减压术植入外泌体三维仿生支架，通过MicroCT、组织学分析及免疫组化验证其体内修复效果，并结合高通量测序与生物信息学分析探讨其修复机制。

研究结果

通过双法连提技术，本研究成功获得了粒径30–150 nm、具有完整的双层膜结构、高表达外泌体标志物（CD9、CD63、CD81）的高质量外泌体。制备的BMSCs-exos@nHA/Col-Ti仿生支架具有317 ± 42 μm的孔径、78.61 ± 2.8%的孔隙率、63.74 ± 2.52 MPa的抗压强度及1.32 ± 0.78 GPa的弹性模量，力学性能接近正常骨组织；GelMa水凝胶实现了外泌体缓释，稳定释放可达28 ± 4天。体外实验显示，外泌体仿生支架显著促进BMSCs增殖、迁移和成骨分化，ALP染色和ARS染色分别验证其对早期成骨分化及晚期骨矿化的显著促进作用；qPCR检测显示成骨相关基因（ALP、RUNX2、OC、Col-1）和血管相关基因（VEGF）表达显著上调。小管形成实验表明，支架组形成的血管网络更密集且闭合环数量显著增加。在体内实验中，MicroCT分析显示，外泌体仿生支架组骨小梁厚度（Tb.Th）达到374 μm，数量（Tb.N）为1.432/mm，间距（Tb.Sp）缩小至382 μm，骨量（BV/TV）提升至51.7%，接近健康对照组水平。组织学染色显示支架表面形成大量胶原纤维及矿化层，CD31免疫组化结果提示支架孔隙内血管密度显著提升。此外，高通量测序与生物信息学分析揭示，支架的修复作用主要通过8条lncRNA-mRNA网络实现，顺式调控网络中的LOC100352431-IT1 通过减少骨坏死区域的炎症反应促进修复，反式调控网络中的LOC100349956-OT2 通过抑制病理性基因的表达发挥作用。

结论

本研究构建了一种负载BMSCs外泌体的三维仿生支架，通过多维仿生设计实现力学支撑、成骨诱导及血管再生的协同作用。支架通过髓芯减压通道精准递送外泌体至股骨头坏死区域发挥长效缓释的作用，显著促进骨与血管的协同修复，并通过高通量测序阐明了顺式反式lncRNA-mRNA调控网络在修复过程中的潜在机制。本研究不仅丰富了外泌体在股骨头坏死中的机制研究，也为股骨头坏死早中期保髋治疗提供了新思路。

Background

Osteonecrosis of the femoral head (ONFH) is a progressive condition characterized by bone necrosis due to disrupted blood supply, often leading to joint collapse and necessitating total hip arthroplasty (THA). While core decompression (CD) is a common joint-preserving surgery for early- to mid-stage ONFH, the lack of an optimal implant capable of providing mechanical support and promoting bone regeneration and angiogenesis limits its efficacy. This study aimed to develop a biomimetic scaffold incorporating bone marrow mesenchymal stem cell-derived extracellular vesicles (BMSCs-exos) for enhanced ONFH repair.

Methods

BMSCs-exos were isolated using an optimized protocol involving Percoll gradient centrifugation, ultracentrifugation, and chemical precipitation, yielding high-purity exosomes (30–150 nm) with intact bilayer membranes and elevated expression of CD9, CD63, and CD81. Biomimetic scaffolds (BMSCs-exos@nHA/Col-Ti) were fabricated via 3D printing, freeze-drying, and GelMa hydrogel photopolymerization. The scaffolds' physicochemical and biological properties were assessed using SEM, XRD, mechanical testing, and confocal imaging. In vitro, their effects on BMSCs proliferation, migration, osteogenesis, and angiogenesis were evaluated. And in vivo, rabbit models of steroid-induced ONFH were used to assess the scaffold's therapeutic efficacy via Micro-CT, histology, and bioinformatics analysis.

Results

The exosome-enriched scaffold exhibited a highly porous structure (pore size = 317 ± 42 μm, porosity = 78.61 ± 2.8%) with mechanical properties resembling native bone (compressive strength = 63.74 ± 2.52 MPa, elastic modulus 1.32 ± 0.78 GPa). GelMa hydrogel facilitated sustained exosome release for over 28 ± 4 days. In vitro, the scaffold significantly enhanced BMSCs proliferation, migration, and osteogenic differentiation. ALP and Alizarin Red staining demonstrated robust early and late-stage osteogenesis, while qPCR revealed upregulation of osteogenesis-related genes (ALP, RUNX2, OC, Col-1). Angiogenesis assays showed denser tubular networks and elevated VEGF expression. In vivo, Micro-CT analysis demonstrated improved trabecular architecture, with trabecular thickness (Tb.Th) reaching 374 μm, trabecular number (Tb.N) at 1.432/mm, trabecular spacing (Tb.Sp) reduced to 382 μm, and bone volume fraction (BV/TV) increased to 51.7%, nearing healthy control levels. Histological staining revealed dense collagen deposition (Masson staining), extensive osteoid coverage (methylene blue-acid fuchsin staining), and mineralized layers on the scaffold surface (Von Kossa staining). CD31 immunohistochemistry showed increased vascular density with an interconnected capillary network in scaffold pores. High-throughput sequencing identified lncRNA-mRNA networks as key drivers of repair, with 420 upregulated and 565 downregulated lncRNAs, alongside 311 upregulated and 290 downregulated mRNAs. Notably, LOC100352431-IT1 reduced inflammatory responses in necrotic bone regions via cis-regulation, while LOC100349956-OT2 suppressed pathological gene expression via trans-regulation.

Conclusion

This study developed a biomimetic scaffold enriched with exosomes, offering superior mechanical and biological properties. The scaffold demonstrated robust osteogenesis and angiogenesis in vitro and in vivo, providing an effective solution for post-core decompression repair in ONFH. Mechanistic insights into the lncRNA-mRNA regulatory networks further support its therapeutic potential, offering a promising strategy and theoretical foundation for early- to mid-stage joint-preserving treatment of ONFH.