Engineering Innervated Muscle-Tendon Constructs for Tissue Regeneration

描述（由申请人提供）：终末期器官衰竭或组织损失是医学上最具破坏性和最昂贵的问题之一。具有功能性肌腱（MTJ）和神经肌肉（NMJ）连接的工程肌肉骨骼组织的创建不仅可以恢复创伤性损伤后肌肉、肌腱和神经等复杂组织的功能，而且还可以用作研究发育肌肉生物学和肌肉药理学的模型。我们已经证明，将胎儿神经组织与胎儿或工程肌腱和工程肌肉组织共同培养，可以产生具有可存活的肌肉-肌腱界面的构建物，在力产生过程中保持完整，以及可存活的神经肌肉连接，促进构建物内肌肉组织的表型[1,2]。形成的组织表达新生儿结构，在没有电或机械负荷的情况下，在功能或表型上没有实质性的进步。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结构所需的两个主要组织界面。我们建议研究这些结构的长期可行性；增加神经营养蛋白如神经胶质源性神经营养因子（GDNF）的表达，并引入合成的、工程化的和基于组织的导管来增强肌肉结构的神经支配；使用生物反应器将神经肌腱-肌肉结构置于模拟应力、应变和收缩活动的物理环境中，类似于在体内后肢肌肉中发现的机械环境；并在体内植入结构体，使其与后肢的实际机械和生化环境相结合。我们的工作假设是，NMJs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先进的表型，增加工程肌肉的力量产生，以及更强壮和更发达的组织界面。体外工程构建体的体内植入将进一步促进所有组织和组织界面的表型和功能。本项目将涉及以下具体目标：设计和制造具有功能性mtj和NMJs的神经-肌腱-肌肉构建体，并在三个发育时间点评估构建体的结构和功能完整性。具体目标2。发展导管系统，引导多神经延伸向肌肉构造生长，从而增加肌肉构造的神经支配比例。具体目标3。目的探讨电刺激下肌肉收缩对神经-肌腱-肌肉结构和功能的影响。具体目标目的：评价在体内植入宿主动物4周后对神经-肌肉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.