Non-Invasive, Quantitative, and Label-Free Characterization of Tissue Engineered Skeletal Muscle

随着研究人员寻求将令人兴奋的技术转化为受损组织的再生，缺乏非 工程化组织使用前的侵入性和无菌方法定量评估质量 患者是一个巨大的挑战。获得FDA认证的工程化组织产品相对较少 批准，所以许多翻译技术都没有前置条件作为导航路径的指南 从长椅到床边。拟议的研究旨在验证光学分子成像(OMI)作为一种工具 工程化骨骼肌在制造过程中和制造后的活性特征，并预测 成功的植入也可能对其他组织工程产品具有广泛的适用性。当前 利用侵入性组织学染色或无菌功能测试对工程化骨骼肌进行评估。 这两种方法都不能避免对珍贵样品的不可挽回的损害，同时提供定量的 严格的产品质量测试所需的数据。为了解决这个问题，我们建议使用非线性OMI来 评价组织工程骨骼肌组织的代谢活性和结构完整性 制造工艺。具体来说，内源性黄素腺嘌呤二核苷酸(FAD)的荧光强度 和烟酰胺腺嘌呤二核苷酸(NADH)将通过无标记OMI作为 新陈代谢活性和细胞活力的测量。此外，二次谐波产生特征 由肌肉肌节中的肌球蛋白和细胞外基质中的胶原纤维产生的 与功能结果相关的结构组织的衡量标准。通过将这两个自由标签结合在一起 成像技术，我们将评估工程骨骼肌组织的代谢和结构状态 在制造过程中。这些具有特征性的工程化组织随后将被植入再生的 以检验所提出的成像方法预测损伤修复成功的能力。它是 预计体外评估骨骼肌的黄金标准可以无创地复制和 用这种方法是无菌的。如果成功评估骨骼肌，这项技术也可以应用于其他 工程化组织，允许可靠地评估各种组织工程技术之前 作为一种修复方式。

As researchers seek to translate exciting technologies for the regeneration of damaged tissue, the lack of non- invasive and sterile methods for quantitatively evaluating the quality of engineered tissues prior to use in patients presents a significant challenge. Relatively few engineered tissue products have received FDA approval, so many translational technologies have no antecedent as a guide for navigating the path from bench to bedside. The proposed research intends to validate Optical Molecular Imaging (OMI) as a tool for characterizing the viability of engineered skeletal muscle during and following fabrication, and predict successful implantation that could also have broad applicability to other tissue engineering products. Current evaluation of engineered skeletal muscle utilizes invasive histological staining or non-sterile functional testing. Neither method avoids irrevocable damage of precious samples while simultaneously providing the quantitative data required for rigorous product quality testing. To address this issue, we propose to use nonlinear OMI to evaluate the metabolic activity and structural integrity of engineered skeletal muscle tissues during our tissue fabrication process. Specifically, the fluorescence intensity of endogenous flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NADH) will be detected and quantified through label-free OMI as a measure of metabolic activity and cell viability. Additionally, the second harmonic generation signatures generated by myosin in muscle sarcomeres and collagen fibers in the extracellular matrix will be quantified as a measure of structural organization correlated with functional outcomes. By combining these two label free imaging techniques, we will evaluate the metabolic and structural status of engineered skeletal muscle tissues during fabrication. These characterized engineered tissues will then be implanted into a regenerative environment to examine the ability of the proposed imaging method to predict successful repair of damage. It is expected that the gold standards for evaluating skeletal muscle in vitro can be replicated non-invasively and sterilely by this method. If successful for evaluating skeletal muscle, this technique could be applied to other engineered tissues, allowing for reliable evaluation of a wide variety of tissue engineering technologies prior to use as a repair modality.