Tissue Engineering of axially vascularized skeletal muscle tissue using functional nanoscaffolds in the rat animal model

功能性骨骼肌的创造仍然是组织工程领域的一个主要挑战。除了寻找最优的细胞源外，开发合适的矩阵用于种子细胞的平行和三维排列是至关重要的一步。此外，生长因子在肌肉形成过程中起着重要作用。生长分化因子-11 （GDF-11）以及igf结合蛋白4,5和6 （IGFBP 4,5,6）可能通过不同的作用影响肌源性分化。由于原代成肌细胞在几次传代后就失去了分化能力，因此这些细胞的单独应用不适合产生大型肌肉组织。因此，间充质干细胞因其增殖和分化能力而成为有吸引力的候选者。骨髓源性干细胞（BMSC）与原代成肌细胞共培养已显示出向成肌系分化的能力。对于再生应用来说，更令人兴奋的细胞来源是脂肪来源干细胞（ADSC），它可以在临床环境中通过微创手术获得。具有排列纤维的电纺丝纳米支架材料是一种很有前途的基质，因为它们可以再现机械各向异性，并且可以与ECM聚合物、蛋白质和多糖结合。一个重要的方面是旋转过程本身：考虑到以后从工作台到病床的应用程序，应该避免使用有毒组件。在目前的研究中，已经建立了电纺丝平行排列pcl -胶原i纳米支架上原代成肌细胞与骨髓间充质干细胞的三维共培养。接下来的实验将集中于用临床上更有吸引力的ADSC取代BMSC。最重要的是，新开发的纺丝工艺是基于无毒溶剂醋酸，而不是像HFIP这样的有毒溶剂。这种新工艺的开发，包括对批次间不一致性的标准化评估，一直是一项重大努力，但现在可以为本提案的实验目的旋转大量的纳米支架。由于目前对分化因子（特别是HFG）的研究还很薄弱，因此，对于目前计划和在此申请的部分，我们将通过生物反应器分析GDF-11、IGFBP4、5和6对肌分化的影响，以及不同培养条件（静态和动态）对肌分化的影响。目前，纳米支架是通过整合含有生长因子的纤维来实现功能分化的。此外，牺牲纤维，含有聚乙烯氧化物，应促进支架的血管化在体内应用。最后，将种子支架植入申请人最近建立的大鼠epi -loop模型中，该模型包含一个动静脉环和一个运动神经。该模型使未来的血管化和神经化的工程新肌肉组织。

The creation of functional skeletal muscle is still a major challenge in the field of tissue engineering. Besides finding the optimal cell source, developing a suitable matrix for parallel and 3D alignment of the seeded cells is crucial step. Furthermore, growth factors play an important role during myogenesis. Growth differentiation factor-11 (GDF-11) as well as IGF-binding proteins 4, 5, and 6 (IGFBP 4, 5, 6) may influence myogenic differentiation via different actions. Since primary myoblasts lose their differentiation capacities after a few passages, the exclusive application of these cells is not suitable for the creation of large muscle tissues. Thus, mesenchymal stem cells are attractive candidates due to their proliferative and differentiation capacities. Bone-marrow derived stem cells (BMSC) in co-culture with primary myoblasts have shown their ability to differentiate into the myogenic line. An even more exciting cell source for regenerative applications are adipose-derived stem cells (ADSC), which can be harvested by minimally invasive procedures in a clinical setting.Electrospun nanoscaffolds with aligned fibers are a promising matrix as they reproduce the mechanical anisotropy and can be combined with ECM polymers, proteins and polysaccharides. An important aspect is the spinning process itself: toxic components should be avoided in light of a later bench-to-bedside application.In the current study the 3D co-culture of primary myoblasts with BMSC on electrospun parallel aligned PCL-collagen I-nanoscaffolds have already been established. Upcoming experiments would be focusing on replacing BMSC by clinically even more attractive ADSC. Most importantly the newly developed spinning process is now being based on the non-toxic solvent acetic acid instead of toxic solvents like HFIP. Development of this new process, including standardized assessment of batch-to-batch inconsistencies, has been a major effort, but now large quantities of nanoscaffolds can be spun for experiments aimed for in this proposal.Since the myogenic impact of differentiation factors investigated so far (in particular HFG) has only been weak, for the currently scheduled and hereby applied part of the proposal, the influence of GDF-11, IGFBP4, 5, and 6 on myogenic differentiation is analyzed as well as the influence of different culture conditions, static vs. dynamic via a bioreactor. Currently, nanoscaffolds are functionalized by integration of fibers containing growth factor aiming at myogenic differentiation. In addition, sacrificial fibers, containing polyethylenoxide, should facilitate the vascularisation of the scaffolds for in vivo application. Finally, the seeded scaffolds will be implanted into the rat EPI-loop-model, established recently by the applicant, containing an arteriovenous loop as well as a motor nerve. This model enables future vascularisation and neurotisation of the engineered neo-muscle tissue.