Engineering Innervated Muscle-Tendon Constructs for Tissue Regeneration

描述(申请人提供)：终末期器官衰竭或组织丢失是医学上最具破坏性和代价最高的问题之一。具有功能性肌腱(MTJ)和神经肌肉(NMJ)连接的工程化肌肉骨骼组织的建立不仅可以恢复创伤后肌肉、肌腱和神经等复杂组织的功能，而且可以作为研究发育肌肉生物学和肌肉药理学的模型。我们已经证明，胎儿神经组织与胎儿或工程化肌腱和工程化肌肉组织的共培养产生了具有可存活的肌肉-肌腱界面的构建物，这些肌腱界面在力量产生过程中保持完好，并且可存活的神经肌肉连接促进了构建物中肌肉组织的表型[1，2]。形成的组织表现出新生儿的结构，在没有电或机械负载的情况下，在功能或表型上不会实质性推进。本研究的目的是设计、制造和评估含有肌腱连接(MTJ)和神经肌肉连接(NMJ)的三维(3-D)工程组织的结构和收缩特性，这是功能性肌肉骨骼构造所需的两个主要组织界面。我们建议研究这些结构的长期生存能力；增加神经营养蛋白如胶质衍生神经营养因子(GDNF)的表达，并引入合成的、工程的和基于组织的管道来增强肌肉结构的神经支配；使用生物反应器将神经-肌腱-肌肉结构放置到物理环境中，模拟体内后肢肌肉中的机械环境中的应力、应变和收缩活动；并将这些结构植入体内，使其周围具有后肢的实际机械和生化环境。我们的工作假设是，NMJ数量的增加与体外施加的电活动相结合，将导致肌肉和肌腱中更高级的表型，工程化肌肉中的力产生增加，以及更强大和更发达的组织界面。体外工程结构的体内植入将进一步提高所有组织和组织界面的表型和功能。本项目将致力于以下具体目标：具体目标1.设计和制造具有功能MTJ和NMJ的神经-肌腱-肌肉结构，并在三个发展时间点评估结构和功能的完整性。具体目的2.发展一种管道系统，引导多个神经延伸向肌肉结构生长，从而提高肌肉结构的神经支配比率。具体目的3.评价电刺激后肌肉收缩对神经-肌腱-肌肉结构和功能的影响。具体目的4.评价体内植入宿主动物对四周后神经-肌腱结构和功能的影响。

DESCRIPTION (provided by applicant): End-stage organ failure or tissue loss is one of the most devastating and costly problems in medicine. The creation of engineered musculoskeletal tissue with functional myotendinous (MTJ) and neuromuscular (NMJ) junctions will not only restore the function of complex tissues such as muscle, tendon, and nerve following traumatic injury, but can also be used as a model for studying developmental muscle biology and muscle pharmacology. We have demonstrated that the co-culture of fetal nerve tissue with fetal or engineered tendon and engineered muscle tissue produces constructs with viable muscle-tendon interfaces that remain intact during force production, and viable neuromuscular junctions that advance the phenotype of the muscle tissue within the construct [1, 2]. The tissues formed express neonatal structures and do not substantially advance functionally or phenotypically in the absence of electrical or mechanical loading. The purpose of this study is to design, fabricate, and evaluate the structural and contractile characteristics of three-dimensional (3-D) engineered tissues containing myotendinous junctions (MTJ) and neuromuscular junctions (NMJ), two of the principal tissue interfaces required for a functional musculoskeletal construct. We propose to study the long-term viability of these constructs; to increase the expression of neurotrophic proteins like glial derived neurotrophic factor (GDNF) and introduce synthetic, engineered, and tissue based conduits to enhance innervation of the muscle constructs; to use bioreactors to place the nerve-tendon- muscle constructs into physical environments which simulate the stress, strain, and contractile activity resembling the mechanical milieu found in hind limb muscles in vivo; and to implant the constructs in vivo to surround the construct with the actual mechanical and biochemical environment of a hindlimb. Our working hypothesis is that an increase in the number of NMJs in conjunction with applied electrical activity in vitro will lead to a more advanced phenotype in the muscle and tendon, increased force production in the engineered muscles, and stronger and more developed tissue interfaces. In vivo implantation of in vitro engineered constructs will further advance the phenotype and functionality of all tissues and tissue interfaces. This project will address the following specific aims: Specific Aim 1. To design and fabricate nerve-tendon-muscle constructs with functional MTJs and NMJs and evaluate both the structural and functional integrity of the constructs at three time points of development. Specific Aim 2. To develop a conduit system to direct the growth of multiple neural extensions towards the muscle construct thus increasing the innervation ratio of the construct. Specific Aim 3. To evaluate the effect of muscle contraction in response to electrical stimulation on the structure and function of the nerve-tendon-muscle construct. Specific Aim 4. To evaluate the effect of an in vivo implantation into a host animal on the structure and function of the nerve-muscle tendon construct at four weeks.