可编程3D神经元芯片平台：整合近实时生物传感与多轴加载技术用于机械生物学损伤分析

本文介绍了一种创新的神经元损伤芯片（NIOC）平台，该平台整合了可编程多轴机械加载与嵌入式电化学生物传感技术，实现了对中枢神经系统（CNS）创伤的精准模拟与近实时监测。该系统通过施加拉伸（extension）、扭转（torsion）及其复合载荷，模拟生理相关损伤条件，并利用高灵敏度传感器动态检测总Tau蛋白（T-Tau）和神经丝轻链（NFL）的释放。研究揭示了载荷特异性生物标志物轨迹与凋亡阈值，为创伤性脑损伤（TBI）的分子机制研究、生物标志物发现及神经保护策略筛选提供了全新工具。

　　

1 引言

中枢神经系统（CNS）对机械扰动高度敏感，易引发创伤性脑损伤（TBI）、脊髓损伤（SCI）或脑震荡。全球每年有超过9000万人遭受CNS损伤，但其分子机制仍不明确。临床诊断主要依赖症状和影像学检查，难以捕捉亚临床或 evolving病理变化，尤其是轻度损伤中影像学正常但症状持续的情况。流体蛋白生物标志物如总Tau蛋白（T-Tau）和神经丝轻链（NFL）在损伤后迅速释放到脑脊液（CSF）和血液中，其水平可反映损伤严重程度并预测长期神经退行性变。电化学免疫传感器等生物传感技术的进步使得这些标志物的高灵敏度检测成为可能，甚至可在接近床旁的环境中实现。然而，由于在患者和动物模型中难以在损伤后几分钟至几小时内进行连续生物流体采样，捕捉载荷依赖的生物标志物释放动力学仍面临挑战。

在细胞和亚细胞水平，机械损伤会破坏神经元稳态和轴突完整性。急性轴突变形导致膜机械性穿孔、离子失衡、钙内流和细胞骨架破坏，进而引发线粒体功能障碍、氧化应激和Caspase激活，最终导致细胞凋亡和渐进性轴突变性。弥漫性轴突损伤（DAI）是TBI的标志性病理改变，其特征是广泛的神经连接中断和长期神经功能缺损，甚至在没有可见病变的情况下也可发生。亚临床或重复性损伤可诱导持续性微结构损伤、胶质细胞激活和病理性蛋白积聚，增加慢性创伤性脑病（CTE）、阿尔茨海默病等相关神经退行性疾病的风险。

机械载荷是神经元损伤结局的关键决定因素，但单轴拉伸、扭转和多轴组合载荷的独特效应在细胞和分子水平上仍未充分表征。在体内，脑组织常经历不规则的多轴应力，如钝性冲击、快速加减速或爆炸波，导致轴突变形和剪切的不均一模式。力的类型和几何形态决定了应变局部化的位置、微管破裂的方式以及细胞骨架和膜结构的重组方式。例如，扭转剪切与弥漫性轴突损伤强烈相关，而拉伸应变可破坏轴突极性和延伸。尽管生物力学模型预测了不同载荷模式的变形阈值，但由于难以捕捉高时间分辨率的生物特征，将这些力与急性分子事件（蛋白质释放、转录变化和细胞凋亡）联系起来的实验证据仍然有限。

传统的体外系统（包括2D拉伸室、压缩装置和剪切流生物反应器）揭示了细胞对机械应激的基本反应，但其平面培养缺乏天然CNS组织的3D结构、基质刚度和复杂的细胞-基质相互作用。此外，大多数系统依赖酶联免疫吸附测定（ELISA）、蛋白质印迹或固定细胞染色等终点检测方法，这些方法需要样本池化、消耗大量试剂且缺乏动态或纵向分析的时间分辨率。球状体和基于水凝胶的神经元构建部分解决了空间组织和机械环境的问题，但很少包含集成传感或可编程加载。最近的器官芯片创新通过结合工程化3D支架、可调机械驱动和可灌注微流体结构克服了许多这些缺点。与动物模型相比，这些微型化系统提供了卓越的时空控制性、可重复性和伦理优势，同时支持具有近实时分析读数的高通量、多重实验。

为满足对机械生物学损伤通路进行生理相关实时监测的需求，本研究开发了一种可编程的3D神经元损伤芯片（NIOC）平台，该平台将多轴加载与片上生物传感能力相结合。

2 结果与讨论

2.1 神经元损伤芯片（NIOC）平台的工程化

圆柱形3D聚合物微流体芯片为重建体外神经元组织和评估对机械应激的反应提供了生理相关的支架。尽管CAD细胞来源于小鼠，但它们表现出神经元表型，并被广泛用于中枢神经系统（CNS）建模。用于NIOC平台开发的CAD细胞被接种在聚多巴胺（PDA）和胶原包被的微通道中，并在血清富集培养基中培养四天，随后在无血清培养基中培养一天以促进轴突延伸。

Bright-field和免疫荧光成像证实了在圆柱形PDMS微流体管内成功建立了具有活力且空间组织化的3D神经元培养。CAD细胞培养5天后表现出强劲的附着和分化， evidenced by strong MAP2 expression (red), indicating dendritic maturation and F-actin staining (green), confirming cytoskeletal organization and spreading along the inner tube wall. Minimal cleaved Caspase-3 signal (purple) was observed, with apoptotic cells accounting for less than 5% of the population, suggesting low baseline cytotoxicity and high viability under static conditions. Bright-field images highlighted dense neuronal networks and extensive neurite outgrowth. Numerous axonal projections extended both radially and longitudinally along the tubular architecture, with random orientation and no imposed alignment, consistent with spontaneous axonal growth in a 3D environment. These data validate the effectiveness of the PDMS-PDA-collagen composite scaffold in supporting neuronal adhesion, viability, and neurite extension.

2.2 通过质谱验证表面稳定性

为确认PDA涂层在机械应力下的化学稳定性，我们在加载后2小时对培养培养基进行了基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）分析。未检测到与多巴胺单体、PDA低聚物或其他降解产物相关的特征峰，证实涂层在整个加载周期中保持完整。这些发现消除了PDA机械分层或生化污染的可能性， reinforced the suitability of this surface chemistry for long-term, real-time biosensing in mechanically active environments.

2.3 可编程机械加载以模拟生物力学创伤

在真实场景中，如运动相关冲击、车辆事故、跌倒或爆炸暴露，大脑不仅承受线性加速力（例如直接平移冲击），还承受复杂的生物力学载荷，包括拉伸（extension）、旋转剪切（torsion）和组合多轴应力。这些力导致神经组织快速变形，尤其是轴突，并且是弥漫性轴突损伤（DAI）的关键促成因素——这是脑震荡和TBI的定义性病理。在这些力中，旋转或扭转载荷尤其与轴突剪切相关，并且被认为比单独的线性力更具破坏性。另一方面，拉伸或单轴拉伸影响膜通透性、细胞骨架动力学和突触信号传递。重要的是，组合拉伸和扭转密切模拟了真实世界创伤事件中遇到的异质性变形，其中组织经常经历同时的拉伸和扭转，如在橄榄球擒抱、伴有头部旋转的跌倒、追尾碰撞（鞭打）和爆炸伤害中所见。

尽管其生理相关性存在，现有的体外平台很少提供明确、可量化的加载方案——更不用说近实时监测神经元反应的能力。为解决这些限制，我们使用3D神经元损伤芯片（NIOC）系统向CAD神经元培养施加精确控制的轴向拉伸（4–20%）、扭转（30°–300°）和组合拉伸-扭转载荷（4–12% + 30°–90°）。这些编程条件故意跨越亚脑震荡、近阈值和超阈值状态，使得能够根据载荷几何形状和强度对细胞反应进行系统分层。选择的应变和扭曲值对应于变形幅度（最大主应变≈0.10–0.25），这些幅度在实验性拉伸研究和经过验证的脑震荡和亚脑震荡TBI的有限元头部模型中一直与轴突损伤相关。

我们的结果显示，暴露于孤立单轴拉伸或扭转的神经元组织表现出强大的结构韧性：MAP2表达保持稳定，Caspase-3激活极微，表明神经元身份保存和低凋亡。这些反应密切 mirror subclinical mild traumatic brain injury (mTBI), where diffuse axonal stress may not produce overt structural lesions yet initiate subtle neurobiological perturbations. Clinically, these findings are significant. In contact sports such as football, soccer, and hockey, repetitive subthreshold head impacts can cumulatively impair neural integrity despite an absence of acute symptoms. Likewise, whiplash injuries from vehicular collisions often involve complex rotational and tensile loads that rarely yield radiologically detectable damage yet contribute to persistent symptoms such as cognitive dysfunction, dizziness, and headache. The inability of current diagnostic tools to detect these low-grade injuries underscores the need for high-resolution, mechanistically informed platforms like NIOC that can delineate injury thresholds with cellular precision.

组合机械载荷的应用——特别是8–12%拉伸与60°–90°扭转——导致神经元凋亡显著增加，Caspase-3阳性细胞从静态条件下的基线～5%上升到20–25%。这一发现证实了神经元对多轴加载的高度敏感性，超出了拉伸或扭转单独效应的影响。值得注意的是，MAP2表达在这些条件下 largely unchanged, indicating that while a subpopulation of neurons underwent apoptotic signaling, others maintained structural integrity and neuronal identity. Data are consistent with neuropathological findings from human TBI and animal models, where rotational and shear strains—common in whiplash injuries, contact sports, and blast exposure—are primary contributors to DAI. Axonal shearing caused by torsional or combined loading is known to initiate cytoskeletal breakdown and apoptotic cascades. Our results align with such pathophysiological observations: apoptosis increased sharply with combined loading, but the persistence of MAP2+ cells suggests that injury was spatially or temporally heterogeneous, as also reported in histological analyses of moderate TBI cases. The non-linear increase in Caspase-3 activation at higher combined loads also supports the presence of a mechanical threshold beyond which neuronal viability declines sharply. Similar threshold behavior has been reported in brain tissue subjected to angular acceleration or high-rate deformation, where exceeding critical strain magnitudes results in irreversible injury. The fact that Caspase-3 activation was minimal under isolated loading but significantly elevated under combined stress reinforces the role of loading geometry in injury severity.

2.4 分子分析揭示载荷特异性神经退行性特征

为进一步研究明确生物力学损伤的机制效应，我们在基因水平量化了关键TBI和神经元标志物表达的变化，并使用免疫染色评估了凋亡。具体而言，我们在施加单轴拉伸、扭转或组合拉伸-扭转载荷后15分钟测量了Mapt（编码Tau）、Gap-43（与轴突再生相关）和Tubb3（神经元身份标志物）的mRNA表达。这些标志物因其在人类TBI和实验模型中的既定临床相关性而被选择。并行地，我们评估了Caspase-3激活以量化神经元凋亡。

Mapt表达在所有机械应激类型中均以载荷依赖性方式增加。在20%拉伸和300°扭转角度下观察到显著上调（p < 0.05），达到相对于静态对照的约2.5–3倍。在组合加载下，除最低条件（4% + 30°和4% + 60°）外，所有条件均显著上调Mapt表达（p < 0.05），最高诱导出现在12% + 90°，与严重机械应激一致。这一结果与先前报道一致，即Tau（由Mapt编码）在轴突变性和神经创伤后迅速释放或上调。Mapt基因表达与生物传感器检测到的T-Tau蛋白水平之间的一致性证实了NIOC平台可靠地捕捉机械损伤的分子相关性。 Notably, Mapt upregulation also mirrors the Caspase-3 trends, suggesting transcriptional and apoptotic responses are linked in the injury cascade.

Gap-43表达——一个既定的轴突生长和再生标志物——在高应变条件下显著上调：20%拉伸、300°扭转以及8%和12%拉伸与90°扭转组合。这些发现表明机械损伤可能触发修复程序以及损伤级联。这种双重反应反映了体内TBI病理生理学，其中存活神经元激活Gap-43作为再生尝试的一部分。有趣的是，Gap-43增加与Caspase-3激活并不严格相关，表明再生和凋亡通路可能响应机械变形的幅度和复杂性而被独立调节。这些数据 contribute to growing evidence that regeneration attempts may begin early, even in parallel with injury-induced apoptosis.

Tubb3表达在所有加载条件下保持稳定，确认了一般神经元身份的保存。它支持了在图和图中观察到的持续MAP2表达的免疫荧光观察，并排除了一般细胞类型丢失作为观察到的Mapt和Gap-43转录变化的主要原因。Tubb3表达的维持提供了内部验证，即施加的机械应激并未全局抑制神经元标志物， reinforcing the specificity of observed gene regulation.

总结所有加载模式下基因表达 fold changes 的综合热图 provided in Figure S9 (Supporting Information). The map illustrates the coordinated upregulation of Mapt and Gap-43 in response to increasing mechanical stress complexity, particularly under combined extension–torsion conditions, while Tubb3 remains relatively stable, reinforcing the specificity of injury-responsive transcriptional dynamics.

cleaved Caspase-3阳性细胞的量化证实了先前观察到的凋亡趋势。单独拉伸加载并未显著增加凋亡，而300°扭转则增加了，将凋亡 fraction提高到≈15%（p < 0.05 vs control）。最显著的凋亡反应在组合拉伸和扭转下观察到，其中Caspase-3阳性细胞达到～25%，与早期IF结果一致。这一数据 reinforces the synergistic impact of multiaxial loading on neuronal death, as previously observed in vivo. Combined mechanical stresses better simulate conditions of falls, whiplash, or blast injuries, where tissues are subjected to rotational and stretching forces simultaneously. The alignment between Caspase-3 activation and Mapt expression further suggests an interdependent relationship between early cytoskeletal disruption and apoptotic signaling, while Gap-43 provides insight into concurrent repair dynamics.

这些结果提供了对图和图中表征的损伤模式的转录组和细胞水平验证。Mapt的上调和在高机械载荷下Caspase-3激活的增加证实了轴突损伤和凋亡启动的载荷敏感性 nature. These responses closely mirror clinical observations where T-Tau levels and apoptotic markers increase following concussive and sub-concussive impacts. The concurrent elevation of Gap-43 highlights the initiation of compensatory repair mechanisms, suggesting that neuronal responses to injury are not unidirectional but instead involve parallel damage and regeneration programs. Importantly, the results reveal that different mechanical loading modes elicit distinct molecular responses. Extension and torsion independently increased Mapt and Gap-43 expression only at their uppermost magnitudes, whereas combined loading consistently triggered significant upregulation of injury markers and apoptosis. This observation supports the hypothesis that the severity of the injury is dictated not only by load magnitude but also by its geometric complexity, which is often underrepresented in traditional 2D in vitro models. Furthermore, the alignment between apoptotic (Caspase-3+) and transcriptional (Mapt, Gap-43) readouts under combined loading underscores the robustness of these markers for stratifying mechanical injury responses in 3D neuronal systems.

2.5 超灵敏生物传感器实现近实时NFL和T-Tau生物标志物检测

扫描电子显微镜（SEM）用于评估金（Au）工作电极（WE）在生物传感器制造过程中表面修饰的形态进展。在其原始状态，Au WE呈现光滑且无特征的拓扑结构。在形成使用烷硫醇化学的自组装单层（SAM）后，通过傅里叶变换红外（FTIR）光谱确认，并在我们最近的工作中详细说明，未观察到表面纹理的明显变化，表明SAM形成了一个保形的、纳米级的层，均匀涂覆金基底。能量色散X射线光谱（EDS）进一步证实了元素组成的变化，指示化学修饰，尽管SEM在此阶段无法解析形貌区别。在用1-乙基-3-(3-二甲基氨基丙基)碳二亚胺（EDC）和N-羟基琥珀酰亚胺（NHS）激活并缀合针对NFL和T-Tau的特异性单克隆抗体后，WE表面呈现出良好分散的球状纳米和微米级结构——在SEM中清晰可见——与成功的抗体固定一致。这提供了免疫功能化的直接结构证据， confirming the biosensor readiness for target-specific binding.

完成的生物传感器使用差分脉冲伏安法（DPV）进行电化学表征，以监测在每个修饰阶段氧化还原探针相互作用的电流响应。电化学系统包括三电极配置：用于识别和信号转导的金WE，用于电荷转移的碳对电极（CE），以及用于稳定电位控制的银/氯化银（Ag/AgCl）参比电极（RE）。DPV在4 mM铁/亚铁氰化物溶液中进行，以测量由表面修饰引起的电子转移阻力。

裸金（Au）电极表现出115 μA的峰值DPV电流，指示快速的电子转移动力学。SAM形成后，峰值电流下降至98 μA，证实了分子屏障的创建，部分限制了氧化还原物种对电极表面的访问。抗体的缀合进一步抑制了电流，对于NFL下降至91.7 μA，对于T-Tau下降至81.6 μA，反映了与抗体层相关的增加的空间位阻和降低的电导率。随后的牛血清白蛋白（BSA）封闭，封端了残余反应位点，导致信号进一步下降至75.7 μA（NFL）和72.2 μA（T-Tau）。最后， upon exposure to 10 ng/mL of the respective target proteins in phosphate buffer saline (PBS), the current dropped significantly—reaching 32.7 μA for NFL and 28.7 μA for T-Tau—confirming antigen–antibody binding and validating signal generation due to specific protein recognition.

为建立校准性能，在PBS和细胞培养基（DMEM with serum）中制备了T-Tau和NFL蛋白的稀释系列，范围从1 μg mL^?1到0.1 pg mL^?1。在两种介质中，传感器均显示剂量依赖性的峰值电流下降，反映了分析物与固定化抗体逐步结合。在PBS中，T-Tau生物传感器显示电流与浓度之间存在强线性关系， described by the equation Y = 7.84X – 7.51 with R² = 0.99. The NFL biosensor followed a similar linear trend, Y = 8.01X – 8.62, also with R² = 0.99. These responses validate the electrochemical mechanism of signal suppression due to increased insulating layers formed by protein binding. When tested in DMEM medium, which contains serum proteins and electrolytes, both biosensors retained excellent linearity. The calibration curve for T-Tau in DMEM was Y = 7.92X – 2.41 (R² = 0.99), while the NFL biosensor showed Y = 7.71X – 1.23 (R² = 0.99). Although the baseline signal was slightly elevated due to matrix complexity, the high correlation and consistent slope demonstrate that the biosensors are highly robust and retain sensitivity and specificity in complex biological fluids. These results are particularly important, as many prior biosensors suffer from matrix interference when transitioning from buffer to serum or media environments.

与先前报道的电化学生物传感器相比，我们的系统实现了飞克/毫升级的灵敏度，远低于血清和CSF中临床报告的最低T-Tau和NFL浓度。重要的是，设计目的并非实现文献中绝对最低的检测限（LoD），而是确保临床相关灵敏度，同时保持宽动态范围以覆盖TBI中早期至晚期生物标志物水平。这种平衡通过优化电极几何形状、探针密度和电化学询问参数实现，使得能够在数量级范围内进行精确量化而无信号饱和。单步功能化策略进一步确保了均匀的探针固定和高可重复性，这在多步方案中通常具有挑战性。校准曲线的斜率在DMEM中仅 marginally attenuated compared to PBS, despite the protein-rich, electrochemically complex background of serum-supplemented media, indicating minimal nonspecific adsorption and signal degradation due to the optimized blocking and surface chemistry. These findings validate the biosensors as robust, reproducible tools for quantitative detection of T-Tau and NFL, with retained performance across matrices, supporting their translational relevance and suitability for near real-time monitoring in cell culture and preclinical injury models.

2.6 有限元建模验证载荷传递和损伤相关性

为验证施加的机械载荷有效传递到PDMS管内的神经元层，我们使用ABAQUS对实验加载条件进行了有限元建模（FEM）模拟。PDMS管被建模为使用Ogden材料模型的非线性超弹性体。使用C3D20H实体单元生成网格，管的底面被完全约束，而顶面通过参考点耦合进行位移控制加载。通过将模型的轴向力响应与实验张力数据匹配，获得了最佳拟合Ogden参数（μ = 19.14 kPa, α = 1.5, D = 3×10^?8 kPa^?1）。

主应力分析显示，在单轴拉伸下，最大第一主应力从4%应变时的≈0.26 MPa增加到16%应变时的≈1.01 MPa，在20%拉伸时达到1.27 MPa。在扭转下，主应力值随旋转角度缩放，从30°时的28.7 kPa增加到300°扭曲时的288 kPa。对于组合拉伸和扭转，最高应力值出现在12% + 90°，产生约787 kPa的应力，这与实验生物标志物和凋亡反应密切平行。有趣的是，中范围加载组合（例如8% + 60°）导致中间主应力（≈525 kPa），与中度Caspase-3激活和基因上调一致。PDMS管在轴向拉伸（4–20%）、扭转（30°–300°）和组合加载下的有限元模拟 reproduced inner-wall maximum principal strains spanning ≈0.10–0.25, consistent with deformation levels linked to axonal injury in published tissue stretch experiments and FE head models of TBI.

这些模拟证实，外部施加的生物力学输入导致可预测且空间解析的内部应力分布，与T-Tau和NFL等损伤生物标志物相关。值得注意的是，第一主应力 emerged as a consistent frame-invariant scalar metric of mechanical insult. A stress-based damage metric (σ?, Euclidean norm of principal stresses) revealed that stress magnitude alone could stratify injury severity across different loading geometries. Cases with σ? > 0.75 MPa consistently aligned with conditions that induced strong apoptotic and biomarker responses, establishing a quantitative framework for linking mechanical stimuli to neuronal fate. The numerical model validated the experimental approach by demonstrating that extension, torsion, and combined mechanical loads were successfully transferred to the neuron-laden surface of the cylindrical construct. This highlights the NIOC platform's reliability in replicating physiologically relevant mechanical environments in vitro, reinforcing its utility for high-fidelity modeling of TBI.

2.7 急性生物标志物轨迹反映转录和凋亡反应

为评估响应机械应激的动态神经元损伤，使用校准的生物传感器在条件培养基中量化了T-Tau和NFL的浓度。这些蛋白质在实验和临床TBI背景下被 well-established 作为轴突破坏和神经元变性的流体相生物标志物。它们在脑脊液（CSF）和血清中的升高与损伤严重程度、临床结局以及向慢性创伤性脑病（CTE）和脑震荡后综合征等慢性神经退行性疾病的进展相关。在机械加载后的多个时间点对培养基进行采样，以跟踪每种蛋白质的急性释放曲线。在所有加载模式中一致，T-Tau和NFL水平在加载应用后15分钟达到峰值，随后在接下来的2小时内逐渐下降。这种早期爆发模式让人联想到临床研究，其中血清T-Tau在损伤后几分钟至几小时内上升，NFL水平从几小时到几天保持升高，取决于损伤严重程度。

重要的是，生物标志物升高的程度与施加的机械载荷的强度和复杂性成比例。对于扭转载荷，蛋白质水平在90°以下保持低位，但在150°以上扭曲时显著增加。在单轴拉伸下，T-Tau浓度在8%应变以上显著增加，而NFL浓度仅在最高测试应变（≥12%）时上升，表明T-Tau可能是轴突应激的更敏感早期指标。这一发现支持了先前的文献，显示扭转或旋转力对轴突尤其具有破坏性，其中剪切变形已知会导致细胞骨架分解和膜通透性变化。趋势也与研究报告一致，即T-Tau与微管损伤和树突串珠相关，而NFL更强烈地与细胞骨架破裂和轴突横断相关。

最显著的生物标志物升高在组合机械加载条件下观察到，特别是在12%拉伸和90°扭转时。在这些情况下，T-Tau和NFL浓度均超过了任何单轴载荷诱导的水平，突出了多轴加载的协同影响。这种效应与早期发现的凋亡活动增加（Caspase-3+）以及损伤响应基因如Mapt和Gap-43的上调一致，表明对复杂生物力学应激的协调细胞反应。蛋白质释放的时间曲线密切镜像了相同条件下捕捉的转录反应。T-Tau分泌与升高的Mapt表达 coinciding, reflecting early microtubule destabilization, while increased NFL levels paralleled Gap-43 activation, indicative of axonal remodeling and injury repair. This alignment between mRNA and protein signals supports the notion that transcriptional priming contributes to downstream protein release following cytoskeletal strain. The simultaneous activation of Caspase-3 further reinforces a phased model of neuronal response—where early cytoskeletal injury leads to molecular disassembly, apoptotic signaling, and attempted structural compensation.

为补充主要动力学和机械载荷响应数据，我们将所有加载模式下T-Tau和NFL的时间轨迹编译成集成比较格式。这种复合表示捕捉了时间依赖动态和载荷特异性生物标志物释放的缩放， reinforcing the distinct sensitivity thresholds and kinetics of each marker under varying mechanical insults.

Notably, the 15-min T-Tau peak under combined loading coincides temporally with the upregulation of Mapt transcripts and supports the hypothesis that transcriptional activation and protein release are tightly coupled during the acute post-injury phase. This observation is in line with neuropathological findings from both rodent and swine models of TBI, where combined mechanical loads result in diffuse axonal injury, subdural hemorrhages, and glial activation not seen in linear loading alone. Human imaging studies using diffusion tensor imaging (DTI) and Susceptibility-Weighted Imaging (SWI) have also confirmed that rotational and oblique loading patterns are associated with greater white matter disruption than linear impact.

基于生物传感器的热图 further illustrates the condition-specific expression profiles of T-Tau and NFL across the full mechanical load matrix. Consistent with the time-course data, protein levels were modest under low-strain torsion or extension, but markedly elevated under high-strain or combined loading. This representation visually emphasizes the sharp biomarker response threshold triggered by multiaxial loading, supporting the use of multiplex biosensors for injury stratification in mechanically dynamic environments.

2.8 转化潜力与未来方向

NIOC平台代表了用于在生理相关机械加载条件下模拟中枢神经系统（CNS）创伤的下一代器官芯片系统。通过整合可编程多轴加载与电化学生物传感器，NIOC能够对轴突损伤生物标志物如总Tau蛋白（T-Tau）和神经丝轻链（NFL）进行近实时、无标记定量，具有皮克级灵敏度。这种能力从根本上将该平台与传统的体外系统区分开来，后者通常依赖静态、终点检测方法，如ELISA、蛋白质印迹或免疫染色——这些方法需要大样本量、池化，并且无法解析损伤响应的时间动态。

与施加单轴应力的传统拉伸或压缩设备不同，NIOC平台提供多轴加载——包括拉伸、扭转及其组合——反映了人类TBI中涉及的复杂力，来自运动冲击、车辆事故和爆炸伤害。圆柱形微流体设计提供均匀应变分布，如通过有限元建模（FEM）确认，实现精确和可重复的载荷传递。这些多轴输入允许根据载荷幅度和几何形状对神经元损伤严重程度进行分层，这是对弥漫性轴突损伤和其他异质性TBI表型建模的关键进展。

关键的是，该平台将电化学生物传感器直接