Colocalization of gene expression and microscale tissue strains in live tendon explants using barcoded biosensors

使用条形码生物传感器对活体肌腱外植体中的基因表达和微型组织菌株进行共定位

• 批准号: 10373315
• 负责人: Spencer Szczesny
• 金额: $ 19.91万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10373315
• 关键词: Address; Algorithms; Bar Codes; Biophysics; Bioreactors; Biosensor; Biotechnology; Calcium; Catabolism; Cell Communication; Cell Nucleus; Cells; Chronic; Collagen Fibril; Degenerative Disorder; Deposition; Development; Devices; Diffuse; Disease; Endocytosis; Environment; Extracellular Matrix; Fatigue; Fatty acid glycerol esters; Fluorescence; Fluorescence Resonance Energy Transfer; Fluorescent in Situ Hybridization; Gene Expression; Genes; Goals; Gold; Image; In Vitro; Inflammatory; Interleukin-1 beta; Knowledge; Label; Maps; Matrix Metalloproteinases; Measurement; Measures; Mechanics; Mediating; Messenger RNA; Microscope; Modeling; Mus; Musculoskeletal; Oligonucleotide Probes; PPAR gamma; PTGS2 gene; Peptide Hydrolases; Persistent pain; Proteins; Protocols documentation; Reporter; Research; Risk Factors; Scheme; Signal Transduction; Spatial Distribution; Stimulus; Structure; Techniques; Technology; Tendinopathy; Tendon structure; Testing; Time; Tissues; Transfection; Transgenic Mice; Weight-Bearing state; achilles tendon; cartilaginous; collagenase 3; cost; cytokine; density; design; experimental study; fluorophore; in vivo; inflammatory marker; innovation; interest; locked nucleic acid; mechanical load; mechanical stimulus; mechanotransduction; nanorod; novel therapeutics; optical spectra; prevent; repaired; response; spatiotemporal; tendon rupture; two-dimensional

Project Summary Tendinopathy is a chronic degenerative disease that occurs in response to tendon overuse (i.e., fatigue loading). In addition to mechanical damage, tendon fatigue loading induces the expression of catabolic proteases, inflammatory cytokines, and the accumulation of abnormal matrix components (e.g., cartilaginous, fat, and calcium deposits), which further weaken the tissue and drive the progression of degeneration. Understanding why tendon cells fail to restore the tissue structure and instead exacerbate degeneration is critical for preventing tendinopathy. The prevailing hypothesis is that the counterproductive cellular response to tendon overuse results from mechanotransduction of altered biophysical stimuli resulting from local tissue damage. However, there is a fundamental lack of knowledge regarding tenocyte mechanotransduction in the native tissue environment and, therefore, whether altered biophysical stimuli are responsible for tendon degeneration. To address this knowledge gap, the objective of this proposal is to develop a live tissue explant model enabling simultaneous spatiotemporal measurement of local mechanical stimuli and cellular gene expression during tendon fatigue loading. By colocalizing the tenocyte response with the mechanical stimuli present in the native cellular microenvironment, we can directly test whether mechanotransduction is responsible for the negative cellular response observed with tendon degeneration. The critical technological obstacle to achieving this goal is the development of a biosensor that can dynamically detect the spatial distribution of cellular gene expression in tendon explants. Endogenous fluorescent reporters are limited by the time and cost of generating a transgenic mouse for specific genes of interest. Alternative biotechnologies require cell transfection, which is inefficient in tissues with a dense extracellular matrix. Therefore, we will investigate whether gold nanorods, which are internalized via endocytosis, can deliver fluorescently labeled oligonucleotide probes to detect cellular gene expression in tendon explants. In Aim 1, we will optimize the design of gold nanorod-locked nucleic acid biosensors and validate their sensitivity and spatial accuracy in measuring mechanosensitive genes in live tendon explants. Additionally, we will establish a barcode strategy with FRET-pair probes and a spectral unmixing algorithm to simultaneously measure up to ten target genes. In Aim 2, we will use this technology to determine whether the degenerative cellular response to tendon fatigue is spatially associated with changes in local mechanical stimuli (i.e., strains). This project is innovative because our results will identify the stimuli mediating the negative cellular response to tendon overuse and help develop novel therapies for preventing degeneration. Additionally, we will establish the ability to measure spatiotemporal distributions of gene expression in live tissue explants with a dense extracellular matrix (e.g., tendon). This technology will be ground- breaking for musculoskeletal research since explant models are commonly used to study musculoskeletal tissues and existing techniques requiring cell transfection are not effective due to the dense extracellular matrix.

项目摘要 肌腱病是一种慢性退行性疾病，它是响应肌腱过度使用（即疲劳负荷）而发生的。 除了机械损伤之外，肌腱疲劳负荷还会引起分解代谢蛋白酶的表达， 炎性细胞因子以及异常基质成分的积累（例如软骨，脂肪和 钙沉积物），这进一步削弱了组织并驱动变性的进展。理解 为什么肌腱细胞无法恢复组织结构，而加剧变性对于防止 肌腱病。主要的假设是对肌腱过度使用结果的适得其反的细胞反应 从局部组织损伤引起的生物物理刺激改变的机械转导。但是，有 关于天然组织中的替纳cy型机械转导的根本性缺乏知识 因此，环境以及生物物理刺激改变是否导致肌腱变性。到 解决这一知识差距，该提案的目的是开发实现的活组织ePplant模型 同时对局部机械刺激和细胞基因表达的时空测量 肌腱疲劳负荷。通过将替域的反应与天然中存在的机械刺激共定位 细胞微环境，我们可以直接测试机械转导的负责是负的 通过肌腱变性观察到细胞反应。实现这一目标的关键技术障碍 目标是可以动态检测细胞基因的空间分布的生物传感器的发展 肌腱外植体中的表达。内源性荧光记者受到产生的时间和成本的限制 转基因小鼠，用于特定感兴趣的基因。替代生物技术需要细胞转染，即 在具有致密的细胞外基质的组织中效率低下。因此，我们将调查金纳米棒是否 通过内吞作用内在化，可以输送荧光标记的寡核苷酸探针以检测细胞 肌腱外植体中的基因表达。在AIM 1中，我们将优化金纳米棒锁的核酸的设计 生物传感器并验证其灵敏度和空间精度在测量现场的机械敏感基因方面 肌腱外植体。此外，我们将建立一个带有fret-pair探针和光谱的条形码策略 将算法交织以同时测量多达十个靶基因。在AIM 2中，我们将使用这项技术来 确定退化性细胞对肌腱疲劳的反应是否在空间上与 局部机械刺激（即菌株）。该项目具有创新性，因为我们的结果将确定刺激 介导对肌腱过度使用的阴性细胞反应，并有助于开发新的疗法以防止 退化。此外，我们将建立测量基因时空分布的能力 具有致密的细胞外基质（例如肌腱）的活组织外植体中的表达。这项技术将是基础的 肌肉骨骼研究破裂，因为外植体模型通常用于研究肌肉骨骼 由于密集的细胞外基质，组织和需要细胞转染的现有技术无效。