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

当研究人员寻求将令人兴奋的技术转化为受损组织的再生时，缺乏非- 用于在使用前定量评价工程化组织质量的侵入性和无菌方法 患者提出了重大挑战。相对较少的工程组织产品已获得FDA 批准，所以许多翻译技术没有先例作为导航路径的指南， 长凳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， 表征工程化骨骼肌在制造期间和之后的活力，并预测 成功植入，也可以广泛适用于其他组织工程产品。电流 工程化骨骼肌的评价利用侵入性组织学染色或非无菌功能测试。 这两种方法都不能避免对珍贵样品造成不可挽回的损害， 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， 评估在我们的组织中工程化骨骼肌组织的代谢活性和结构完整性， 制造工艺。具体地，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 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蛋白质将被定量为 一种与功能成果相关的结构组织的衡量标准。通过结合这两个标签免费 通过成像技术，我们将评估工程化骨骼肌组织的代谢和结构状态 在制造过程中。然后将这些特征化的工程组织植入再生组织中， 环境来检查所提出的成像方法预测损坏的成功修复的能力。是 预期体外评估骨骼肌的金标准可以非侵入性地复制， 通过这种方法无菌。如果成功评估骨骼肌，这项技术可以应用于其他 工程组织，允许可靠的评估各种各样的组织工程技术之前， 用作修复模式。

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.