研究背景：小耳畸形是一种常见的先天性耳廓发育异常，表现为单侧或双侧耳廓发育不全，给患者带来显著的生理及心理负担。耳廓作为重要的面部轮廓结构，其形态完整性对外观美感及听力功能均有重要意义。目前，小耳畸形的临床治疗方法主要包括自体肋软骨移植、人工材料支架重建以及组织工程耳再造等。其中，自体肋软骨重建虽然广泛应用，但存在供区损伤、术式复杂、软骨量受限等局限；人工合成材料如 Medpor虽可提供良好支撑性，但易发生异物反应和植入物排斥，长期稳定性难以保障。因此，发展替代性、高生物相容性的重建策略成为临床迫切需求。近年来，组织工程技术为耳郭再造提供了新思路。通过种子细胞、生物支架和诱导因子构建组织工程软骨块，可实现“自体来源”、“体外预构建”、“精准成型”的理想重建。然而，尽管组织工程软骨在形态构建上取得一定突破，但其在临床应用中仍面临植入后宿主免疫反应显著的问题，表现为炎性细胞浸润、巨噬细胞活化以及成纤维细胞增殖等，最终导致支架被纤维化包绕，阻碍软骨细胞存活和基质沉积，影响组织整合与重建质量。异物引起的慢性炎症反应已成为制约组织工程耳临床转化的重要瓶颈。已有研究显示，巨噬细胞在植入物相关免疫反应中处于中心地位，其数量、活性及分化状态直接影响组织修复的方向。其中，CSF-1（巨噬细胞集落刺激因子-1）及其受体 CSF-1R 在维持巨噬细胞存活与极化中具有关键作用，过度激活的 CSF-1/CSF-1R 信号轴可促进巨噬细胞持续偏向 M2 表型，并驱动纤维化反应。因此，靶向 CSF-1R 信号通路可能成为缓解异物反应、改善免疫微环境、提升组织工程植入效果的有效策略。静电纺丝作为一种高效的纳米支架构建技术，可将小分子药物均匀负载于纳米纤维中，实现局部缓释调控，为开发“材料+免疫调控”一体化植入系统提供了可行途径。

研究目的：本 研 究 旨 在 通 过 静 电 纺 丝 技 术 构 建 负 载 CSF-1R 小 分 子 抑 制 剂（PLX3397/GW2580）的聚己内酯（PCL）纳米纤维支架，利用其局部缓释特性调控植入部位的巨噬细胞反应，减轻炎症和纤维化，营造有利于软骨再生的免疫微环境。具体目标包括：1. 构建负载不同浓度 PLX3397/GW2580 的静电纺丝支架，系统表征其理化性能及药物释放特性；2. 探讨该支架在动物体内植入后对免疫反应、巨噬细胞浸润、炎症因子表达和纤维囊形成的调控作用；3. 进一步联合组织工程软骨块植入动物体内，验证药物支架有利于软骨细胞生存、基质沉积及再生修复方面的效果，明确其在组织工程耳应用中的潜力。

研究方法：1. 药物支架的制备与性能评价：采用共混静电纺丝技术构建 PLX3397/GW2580 三种不同质量浓度（10%、20%、40%）的药物负载型 PCL 纤维支架，并设置空白对照组。对各组支架的形貌结构、纤维直径、热性能、药物释放曲线、降解行为以及细胞相容性进行系统表征，评估其理化性质与生物学性能。2. 药物支架的体内抗炎性能评价：将不同组别支架植入大鼠背部皮下，于术后第 3天、1 周、2 周、4 周和 8 周分别取材，通过 HE 染色、Masson 染色及 COL1A1免疫组化分析纤维囊形成与胶原沉积情况；采用CD68免疫组化、CD206和CSF-1R免疫荧光染色，结合 iNOS 与 CD206 基因表达水平评估巨噬细胞的分布、数量及表型极化状态；并通过 qPCR 检测 IL-6、TNF-α 等炎症因子的表达水平，明确药物在调控免疫反应过程中的作用及其浓度依赖性。3. 药物支架与组织工程软骨联合植入的功能验证：提取兔耳原代软骨细胞，构建GelMA 水凝胶软骨块，并分别与不同组别的药物支架包裹后植入兔背部皮下。术后第28天取材，通过HE染色与Masson染色评价纤维化程度，利用CD206、CSF-1R免疫荧光以及 iNOS、CD206、IL-6、TNF-α 基因表达评估局部炎症反应情况；同时通过阿利新蓝染色检测软骨基质沉积情况，并测定 GAG 含量及软骨相关基因（SOX9、ACAN）表达水平，从而评价各组在软骨再生方面的效果差异。

研究结果：1. 成功构建了 PLX3397/GW2580 三种浓度（10%、20%、40%）的药物负载 PCL 静电纺丝支架，并明确其理化性能与生物相容性：所有支架均表现出良好的纤维结构和稳定性，纤维排列致密，直径均匀；药物负载后支架的降解速率均有提升。药物释放曲线显示，PLX3397/GW2580 支架均呈现初期突释加持续缓释的双相释放模式，浓度越高释放速率越快。CCK-8 细胞增殖实验显示，高浓度药物支架在早期对细胞增殖有轻度抑制，但随药物释放浓度降低，细胞活性逐渐恢复，支架整体具有良好生物相容性。2. 药物支架在动物体内显著缓解了植入诱导的免疫炎症反应并抑制纤维化形成：通过大鼠皮下植入实验观察到，PLX3397/GW2580 支架均有效降低局部 CD68+巨噬细胞数量、抑制 M2 型极化，CSF-1R 表达下调；炎症因子（IL-6、TNF-α）表达在多个时间点明显下降，支架周围纤维囊厚度显著减少，Masson 染色与 COL1A1染色显示胶原沉积受限，体现出明确抗纤维化效果。其中，PLX3397 组在早期巨噬细胞浸润和 IL-6 表达的抑制方面表现更强，而 GW2580 组在 M2 型极化标志物（CD206）表达方面下降更显著。药物对炎症调控存在一定浓度依赖性，40%组效果最为突出。3. 药物支架与组织工程软骨联合植入兔体内后有利于软骨再生：与未载药的 PCL 纺丝组及无纺丝包裹的纯 GelMA 组相比，载药支架组的炎性细胞浸润明显减少，局部纤维化程度显著缓解，CD206 与 CSF-1R 的表达显著降低。软骨再生评价显示，载药组阿利新蓝染色更为明显，GAG 含量提升，SOX9 和 ACAN 的基因表达水平亦显著上调，提示软骨基质沉积更丰富，结构更加完整。PLX3397 与 GW2580 两种药物均表现出有利于软骨再生的优势，其中 GW2580 的效果略优于 PLX3397，这可能与 GW2580 更显著地抑制巨噬细胞向 M2 型极化并提供更有效的基质保护作用相关。

研究结论：本研究采用共混静电纺丝技术成功构建了具有良好生物相容性的可双相释放PLX3397/GW2580 的 PCL 药物支架，载药支架在体内植入后可有效抑制巨噬细胞浸润及炎症因子表达，显著降低纤维囊形成；与组织工程软骨块联合使用时，载药支架可优化植入微环境，有利于软骨基质生成，提升软骨再生质量。本研究为将免疫调控策略引入组织工程耳再造提供了新思路，为解决异物反应与再生失败难题奠定了理论和实验基础。

Background： Microtia is a common congenital auricular malformation characterized by unilateral or bilateral underdevelopment of the external ear, resulting in significant physical and psychological burdens for affected individuals. In China, the incidence is estimated to be 3-5 per 10,000 newborns, with regional variations. As a critical component of facial contour,the auricle plays an important role in both aesthetics and auditory function. Currently, clinical treatments for microtia include autologous costal cartilage reconstruction, synthetic implant-based reconstruction (e.g., Medpor), and tissue-engineered auricular reconstruction. Among these, autologous costal cartilage is widely used due to its biocompatibility, but it has limitations such as donor site morbidity, limited cartilage volume, and surgical complexity. Synthetic implants like Medpor offer structural support but are prone to foreign body reactions (FBR) and implant rejection, and their long-term stability remains uncertain. Therefore, there is a pressing need for alternative reconstruction strategies with better biocompatibility. In recent years, tissue engineering has offered a new avenue for auricular reconstruction. By combining seed cells, bioactive scaffolds, and inductive factors, tissue-engineered cartilage constructs can be developed with the potential for autologous origin, pre-fabrication, and anatomical precision. However, despite progress in shaping tissue-engineered cartilage, its clinical translation remains hindered by pronounced host immune responses after implantation, including inflammatory cell infiltration, macrophage activation, and fibroblast proliferation. These responses lead to fibrotic encapsulation of the scaffold, impairing chondrocyte survival and extracellular matrix (ECM) deposition, ultimately compromising tissue integration and regeneration quality. Chronic inflammation caused by foreign body reactions has become a major bottleneck in the clinical translation of engineered auricular tissues. Macrophages play a central role in the host response to implants. Their abundance, activation state, and polarization profile determine the trajectory of tissue remodeling. Among the regulatory pathways, colony-stimulating factor 1 (CSF-1) and its receptor (CSF-1R) are essential for macrophage survival and phenotype maintenance. Persistent activation of the CSF-1/CSF-1R axis promotes M2-like polarization and drives fibrotic responses. Thus, targeting the CSF-1R signaling pathway is a promising strategy to mitigate foreign body reactions, improve the immune microenvironment, and enhance implant integration. Electrospinning, as a versatile nanofiber fabrication technique, enables the  uniform incorporation of small-molecule drugs into polymer fibers and allows for localized, sustained drug release, offering a practical approach for developing scaffolds with built-in immunomodulatory capabilities.

Objective： This study aims to construct polycaprolactone (PCL)-based electrospun nanofiber scaffolds loaded with CSF-1R small-molecule inhibitors (PLX3397 and GW2580) and to investigate their effects on modulating macrophage responses at the implantation site. The ultimate goal is to attenuate inflammation and fibrosis, thus creating a favorable immune microenvironment for cartilage regeneration. Specific objectives include: 1. Fabricate electrospun scaffolds incorporating PLX3397 and GW2580 at three different concentrations (10%, 20%, 40%), and characterize their physicochemical properties and drug release profiles. 2. Evaluate the in vivo effects of drug-loaded scaffolds on immune response, macrophage infiltration, inflammatory cytokine expression, and fibrous capsule formation. 3. Combine the drug-loaded scaffolds with engineered cartilage constructs and assess their efficacy in supporting chondrocyte survival, ECM deposition, and overall regeneration in vivo, establishing their potential for application in tissue-engineered auricular reconstruction.

Methods： 1. Fabrication and Characterization of Drug-Loaded Scaffolds: Electrospun PCL scaffolds loaded with PLX3397 or GW2580 at concentrations of 10%, 20%, and 40%(w/w) were prepared via solution blending, with a pure PCL group as control. The morphology, fiber diameter, thermal properties, drug release kinetics, degradation behavior, and cytocompatibility of each group were systematically evaluated to assess their physical and biological performance. 2. In Vivo Evaluation of Anti-Inflammatory Effects: Scaffolds from each group were implanted subcutaneously in rats. Tissues were harvested on days 3, 7, 14, 28, and 56 post-implantation. Hematoxylin-eosin (HE), Masson's trichrome, and COL1A1 immunohistochemistry were performed to evaluate fibrous capsule formation and collagen deposition. CD68 immunohistochemistry and CD206/CSF-1R immunofluorescence staining, combined with gene expression analysis of iNOS and CD206, were used to assess macrophage infiltration, quantity, and polarization. Additionally, qPCR was performed to detect the expression of inflammatory cytokines IL-6 and TNF-α, clarifying the dose-dependent effects of the drugs on immune  regulation. 3. Functional Verification with Engineered Cartilage Constructs: Primary chondrocytes were isolated from rabbit auricular cartilage and used to fabricate GelMA-based hydrogel cartilage constructs. These constructs were wrapped with the respective scaffolds and implanted subcutaneously in rabbits. After 28 days, samples were collected for histological analysis. HE and Masson staining assessed fibrotic responses; immunofluorescence for CD206 and CSF-1R, along with gene expression of iNOS, CD206, IL-6, and TNF-α, evaluated local inflammation. Alcian blue staining, GAG quantification, and SOX9/ACAN gene expression analysis were used to assess ECM production and chondrogenic outcomes across different groups.

Results： 1. Successfully fabricated PCL electrospun scaffolds loaded with PLX3397/GW2580 at concentrations of 10%, 20%, and 40%, demonstrating stable fibrous structures with uniform fiber diameter. Drug loading accelerated scaffold degradation and exhibited a biphasic drug release pattern, initially rapid and subsequently sustained. CCK-8 assays indicated high concentration groups slightly inhibited initial cell proliferation, but cellular viability recovered as drug concentration decreased, indicating overall excellent biocompatibility. 2. Drug-loaded scaffolds significantly alleviated inflammatory responses and fibrotic encapsulation post-implantation, effectively reducing local CD68+ macrophages, inhibiting M2 polarization, downregulating CSF-1R expression, and decreasing IL-6 and TNF-α expression at multiple time points. Masson and COL1A1 staining confirmed reduced fibrous capsule thickness and collagen deposition. PLX3397 exhibited stronger early macrophage infiltration and IL-6 suppression, whereas GW2580 more significantly reduced CD206 expression. Effects exhibited concentration dependency, with 40% being most effective. 3. Drug-loaded scaffolds combined with engineered cartilage significantly enhanced regeneration in vivo, reducing inflammatory infiltration, fibrosis, and expression of CD206 and CSF-1R compared to controls. Cartilage regeneration indicators (Alcian blue staining, GAG content, SOX9, ACAN expression) were superior, indicating richer matrix deposition and structural integrity. Both drugs benefit regeneration, with GW2580 slightly superior due to its stronger inhibition of M2 polarization and better matrix protection.

Conclusion： This study successfully fabricated PCL-based electrospun scaffolds capable of biphasic release of CSF-1R inhibitors PLX3397 and GW2580. These drug-loaded scaffolds demonstrated favorable physicochemical properties and biocompatibility. In vivo implantation effectively suppressed macrophage infiltration and inflammatory cytokine expression while significantly reducing fibrous capsule formation. When combined with engineered cartilage constructs, the scaffolds modulated the immune microenvironment, enhanced ECM deposition, and benifited cartilage regeneration outcomes. This study provides a novel immunoregulatory strategy for tissue-engineered auricular reconstruction and lays a theoretical and experimental foundation for overcoming foreign body responses and regenerative failure.